For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. PN 101-855-400 Version 03 (April 2020)

HiFi Library Preparation Using SMRTbell

Express Template Prep Kit 2.0 For De Novo

Assembly and Variant Detection ApplicationsSequel II System v8.0 / Sequel II Chemistry 2.0 / SMRT Link v8.0

HiFi SMRTbell Library Preparation Using SMRTbell

Express Template Prep Kit 2.0 For De Novo Assembly

and Variant Detection Applications

1. HiFi Sequencing Features & Applications Overview

2. HiFi Library Sample Preparation Workflow Details

3. HiFi Library Sequencing Workflow Details

4. HiFi Library Sequencing Performance Example Data

5. HiFi Sequencing Data Analysis Recommendations for Variant Detection and

de novo Assembly Applications

6. Technical Documentation & Applications Support Resources

7. Appendix

i. Optional Alternative Shearing Method (Covaris g-Tubes)

ii. Optional Alternative Size-Selection Method Using the Sage Science BluePippin System for

Variant Detection Applications

iii. Optional Alternative Size-Selection Method Using AMPure PB Size-Selection for De Novo

Assembly Applications

iv. General Recommendations for High-Molecular Weight gDNA Isolation, QC & Handling for

SMRTbell Library Construction

HiFi Sequencing Features & Applications

Overview

HiFi Sequencing Workflow Provides High Accuracy and Long Read Lengths

HIFI READS: A NEW PARADIGM IN DNA SEQUENCING

- Generate higher quality de novo genome

assemblies using a single technology

- Call all variant types – from single nucleotides

to structural variants

- Phase allele-specific haplotypes

- Identify full-length isoform transcripts – no

assembly required

- Comprehensively characterize samples with

complex variation (e.g., bacterial, viral and

cancer cell populations) to explore unique

and evolving genomes.

GENERATION OF HIFI READS BY CIRCULAR CONSENSUS

SEQUENCING (CCS)

Insert sizes up to ~25 kb

(>99% accuracy)

Longer reads allow for multiple passes of the same DNA molecule

~300-600 Gb Raw Bases / SMRT Cell 8M

~80-125 kb average RL

~1 – 4 M HiFi Reads / SMRT Cell 8M

~20 – 40 Gb HiFi Bases / SMRT Cell 8M

Average HiFi Read Accuracy: >99.9% (>Q30)

Up to ~25 kb insert size

GENERATION OF HIFI READS BY CIRCULAR CONSENSUS

SEQUENCING (CCS) (CONT.)

19,820 BP HIFI READ, PREDICTED QV: 33

>m64089_191020_002935/346/ccsGAGTCAACCGCTCTACGCACTGACTCTCGTAAGAATGAGCTTCGAGAACAAAACAAATAAAAAGAGACAGACAGCAAATTTAAAATCGGAAACCAGGAGAAAAAATGGCCAGAGAAGAGCGCAATGACAGCAACGAGGCGCTAATATAGCTATGGCGCCTCGTTACAACGTGAACTCTCCGAAATCGTCTGTTGTTCTTTTTAAAATAGTTGTTATTACATAAATAAGCGGTCGATGCCTAATACCCATTGATTTTGTAGTAAATACA

TATAATACTCGTATTATGAGTATTTACTACACAATATTTTCAACGACATGGCCGTCATTTTAATTCCGTGTCTTGGATTTATGACGTCATAAATATGATGAAAAGATATCGCTGGGTATTCCATGATTAGATTTTATGTGCGCTTTTATGATCCAAGACTCCAATAACTTCGAAGAATTCTTAGTAGAATCTAGTTATTATGAGATTAAAATAAAACTAGATAAACAACTAAGTTGTTTGAAATAATTAATTTTATTCAGCAATTTTT

GGCTTTCCAATTCAATTAGGTGCAGCCTATTAAAAACTAAGTTGCAGTACGCCACCACTTTTAGTTTTTCACAGTCTATTCCTATTATAAAGAACAAGCTGTGAATAAATTACAGTAGTTTCCCTTTCCTAAACTGAAAATACACACAATTATAATCTTATGAAAAACATAAATTCGTTTCATAAAGCTTATCGCTTTGTTTCTTTTTAGAAAATTCTCGGGCTACTTAGACCTCGGCGATGTACTCAGTACCTACCCAAATTGCTGG

ACTGGACTGCGCGATGCAATGTTCTGCGCATGTCCGTTTATATATAGGTAGCCTATTGATATTTTGTTATGACTTTCGTATGTCCTGGAGTAGCGTCCCTAGCTACACCGGCTTTTGCAGTTTTATGCACTCCTGGTAGTATATTCCAAAAAACATGTGACTGCGCCGTGCGTCAGATCGTTATGAAAGCTGATAAGCGACTGAGCGAATATGTCATTATTAAAGAAACTCCGCCATTTTGCTGCCGCCTGCTAACACTATGTGCAAT

GTGCATTCTTCTTCGATTTTCCGATTTTCATTTATGAGTGTAAAATTTTTATTTTGCGATGTAATGAAACTTCTTAATTAATTGAAATTAAACATATGTATAAAGCTGGGGAAGCTCCGTGGCGTCGTGGTTTGAACGATTTGGTTTTGATGTTAAAAAAAGCTGTTTATTTTTACATATTTTTTTATGGTATGGTGAACAATGAAGAGTTATCTATCCATCTATCTTACTTTCCCCGTGTTTAGACTTCGGAGGAACCGGTGCAACG

ATATTTACAATCATTGAAACACATTTAATACAAAAAAAAGTTCTTAAAAGAGACATTTCCTATAGGACATTTCCTGAAATATTTTGTGTAACACTAATGTTTTTATTATGCAACACAATACCTTAGCTACATATTATAAAAAAACTCACAGATATTGTATATATTTGTCTTTTACCTTATATAACGCATCTGTTTGGATTTGTACTGAAGCCATGCGTGCCAACTTTTTAGCATCAACGGTGACATGACAGCCCTTGACAGCCGTTTT

TAACGATGAAATAAAGGAAAGAAAAATTTTAATTTTATTGTGTTATGAGTTGTTATGGGCTTAAAAAACATATTATTTTTAATGGTGACGGTTAACAGCAATGAGTCATTTGGGACCCCATAAAAAAGGGTCAACTAGCCTATTAGAGTTTAATAAGATTAGTCTTGTGGTCGACAGTAATCCAAAAATACCAAAAATACAGTCCGCGTCCTAGGAATCTAGGAATTTATGTATTCAACATTAAAGGCGGATCACTACACGCCTCGAC

CCGTAAATGTGTAGCCACCGTAATGTTTGTATTTAAACACTTCGTCACATTAGAAAATAAAATAAAATAAATTGCCTATTTATTCGTATAAACAAACACATATTATTATATGTAAAATTTAAATAAAATTGCGATCATTAAAAGTTGTGTTATTGACTAGTTCCACGCAGACCAATGGTAGCGATGTTTTTCTCATATCTGCGCATGCCTGATGAGTAACAAATCAGAGGCCAATATTTACTGTTATTTATACGTTATGAATGGGAGC

GTCCGTAGCAACTGCACTTCCTTCAATACTGTGCATTCCTGGTAGTTGATGGACAAAGGCAACGTGGTTTGCACACTGGGTCACGCCCGAAGAACTATTTTATTATTTGTCCGCTGTTGAATTGGTCAGATTGTTTTATTTAGAACAAACGATCCTGTTGCCATGCACCGGGCAAAGCGTAAATTAATCAATTAGTTTTCAATTTTTTTTTCACAGGAAAAAAAATTAAAAACTATTGATTAATTACAATTTAATCTCTAACGACGTC

ATATTGATTTATTGATTCCATACTTTGTAAAACATTAAGTTAGTGTGTGTAACTTCTGCAAAGACAAAAAATAATAAATAAAAAATAAAATTCGAACTTCGAATGTAGATGGTGCACGTTTTTCTTTGGTTTCGGCCGGCCGGCGTCCGTAACAACAGAACCTACTTCTTGTACACTATGAGTCCCTGGTACCCCATCGTATAGAGAACGTGACTTAGACGGGGTTCCAAGTTTTCAACAATGTTTGCTTATTTGTTTATTTATTTAT

TTATTTGCATGTCACATAATCACTGTACAGAAATACAAAGGTCTTAAAAAATAGGATTGTATACATGTGACACCCTGCAAGGGTATAGCAATATATAATTATAATATTGGACCAGAGGGCCCTAGTTACTTAACAATTATATAAACTTATTTTTAAATTAAAATTTTATAAGCAACGTAAAATAAATTCTAATACTCGTATTACTAACAAAATTTAACAGAAACAAGAAAAACATGAGATCAAATTTTAAATTATTTACTTATATACG

TAACGTTATCTACCTTTTAATTACAAAAATTATTATATAGTGGAAGTTATTCGTTTTCGAAAAATTCCTTTAAACTGTAAAAACATTTCTCGATCAAGAACTTTTTTAAGTGCTCAGTAAAATTCGGGAGTTTCTCAAAGTTTTTGATGTCGTTTGGGATATGGTTATATATTTTTATAGACATAAAATATGGACTAGATGAGACTAATTTTAATTTTGTAAAAGGAATTTTAAGTTTATTTAAATATCTGTCATTTCTTTTGATTTT

ATTATTTGGTGAGAAAAATTCTTGATGTTTTCTGGCAAATTTACATGCTTCTAATATGTAAATGGATGTGAGTGTTAATATTCTGTGTTTGATAAAGTGAGGTCTGCATGATTCCATTTGTTCTATATTTGTCAATATTCTCAAACATCTCTTCTGCAATATAAATAATTTATCTATTTCAGAGGAGTTACCCCATAAAACGACGCCATAGGAAAGAATGGAGTATGCATAAGCATAGTATGCTGAGAGAGCTGTTTGAAAGTTTGTA

ACGCGTTTCAAGTGGTGTAGTGCATATATGAAAGATGATAATTTATGTTCTACTTTTTTAATATGCTGCTTCCAGTTTAAATGTGAATCTAGTTCAATGCCCAACAGGGTTGCTGTGTTCACTGTTTCTAATTTTATATTTTTATAACAATATTGTACCTGTAATGAACTTTTTTGATAGGGCTTAAATTGGATTAATTTGGTTTTTTTAAGGTTTAGTTGGAGATTATGTGTTTCTAGCCAGTTCGTAATGTTGTCTAAAATAAAGT

TTAATTTAGTTTTAAGTTCCGATGAGTTTGAGCAAGAGATAAGCACTGAGATGTCATCAGCAAACAATACACACAGATTATCAAAAATATCAGGTAGATTATTTATATAAATGAGGAATAAGACACATCCTAGAACGCTACCTTGAGGTATTGATCCGGTAACTTGGATGGCATTAGATCTGGTGTATGTGATCATTCCTGTCTCAAAGTCTGTATTTTGTATTTCAACGTATTTGGAATTTGGAATTATTTAGTAGTATTTGGAAGT

ATTTGGAATTGTTGAAACCAATAAATAAAACTTATAAAATCCTTATTCTACTTTCTACCATTTCGTGAAACAAACAGTAGATGGCCGATGTTTTTGTGTTCTAGGCCCACTTACAGGAAAATTAATGGAGATTACATAGTAGATATTATATCTAGACTAACCCCCTTACTAATAAAATAATAAAAATAACCGACATCCACGTGGGCGGAGCCACGGGCGACCGCAAGTTTCTTATAAATCTGTTGAAAATTTACGTCAAATGTGAAAA

TTCATGGTCTTTCTTTTATTTCTGATATTATGATGTTATTTAGTAAAGAATAATCCTCACGATGAAACCTTTCGATCGGTATGATAAAGCTACAGGTTAATTATTTTTCTCGTGTAACGGGTTCGATTCCCGGTTCAACAATGTAATTATTTAAATATCTATGAATGCAGTTTAAATAGTTTCTGAAATTGAAATAAAGTCTTCTTTCAGTAAAACGTTCTAACTGTAATATTTAAGGTACTTTCTGTGGTACTTTCCCTTCATGTGG

CATAGCCGTGGGACGGCCTGTATATTGCTGCGTAAATAAATATCTTGCTGCCCAATTAATTATTGTGCTGATCAAATAATAATGTTGCTGCCCAAATAATTATTATGCCGCCCAAATAATTATAATGCTGACAAAGTATTTGTTTGCTGCACAAATTATTTTTCGGGTTACCTTTTTGTTGCTGACCAAATAATTAAGAGACTGCCTAATTAATTACTTCCCGTTTAAAAAACCTCGGAGTAAACAGAACAAGTCCTACAAAATTGTA

TTGAACGTTCATTTTCGATTCACATTAAAGCAAAGGGGCGTTCCACTAAAAATAAAAGTGTCTACGTGATTAACTAGATGGCGCTGTCGTACACCTAAATCGTGCTATAATTTTCTCTCCTATTCTATACAATTTTCAGTTGTTGTATCATGTAAATACCCTGGTCATAAAGTATATTGAAAAAGACTATTTGTTTTATAATTTTTCACTAGATCCTCAATAAAACACCAATGGGTTGTTTAATATTAAAAATATTTACTAATAGTTG

TGAACATAAGAGCTTCCATTCCAGATTAACTACAAACATACTCTTGCAAATATGGGATCCTGGTAGCTCTCCGCAGTTTGTCAATGTGACAAAATGAGGACACTAGTTTGTAGTTTCATTGTTTCCTAGTTTATTAGGCCACGCCTAATCGTATGCCATGCCACTCCACGTACGCAGTCATTGATAACCGTCTGGTTTCGATAATGAAAACAAGGATACTATTATTTTGGATGGACCTCGGATGGATGACCAAAACTTTGTGATTTCG

ACTTCCACTGTGCTTTGGAAGCTGTTGATCTCGGTGTGTCCTGTAGGATGTCTGCTTTTTAATGTGTAATGTGAAATATAGAAACTTCGTTAGAAAACTACACAATAGAATATTGTTGACAAGAGGCTATAGAAACCTTTAAAAAGTCAAATTATTTTTAAGATAACCATAATAAGACTTTAAAAAGTTTGTACATATACGTTTCTATTCACCTGTGTCTACCCCTTCGGCGATAGAGGCGTGATTTTTATATGTTATGTTTATATTT

ATCCTTTCTATTCTACATCTCCGTATCTTCTAAAAATGAAAAGTAATTTGAATATTGAATTTTCTGATCATTGATATGAGTAAGGAGGCTTAAGGGTTTGCATAAAAAAATAATATGAATAAAGTATTATCAAAGTAAATAAACCAAGAAGTAAATCTGGCTTTTCTCTATAATTCCTTAGCTCCGTTTGATTTTCCTATTAAAATAATTAGCAGTGTAAATTGAAAGAAACTCGATAATTACATGCTACTGACAGGGTGGGAGATCG

TTAATCAAATCGCAAGAGAGGGAGCCGGAAGACGTATTTCTCTCGCTTCCACTCATCACTACCTCCATTAACTTTCTCTTTCACTCACAAAATTCGTCGCATGCAATAACAGATTCACATTTTTCACAAATCTCTAAGTTTTAGAGTTTTTTTACTTTAAATTTTCCACGTCTAGTTTTAAAGTGTGGTTTTGTATAACATGAAATAAATATGAAAGCACTAGAGATAGCCTTATGAAAAGAATATATTCGTTGCTGCAGTGAAAATT

GAACCATAACGTAATGGCTGTACATTGTATTTCGTTTGCGTGATAGGCGCAGTGTGCTTCGTAGTTAACTCGTGATGTTGCAGTGCAGTGTTCTATTCTATTGTGGTAAGGGTGCAATTTGCTTGTTCCTGCAATATGCGTGTCTTCTTGTGGGTTATAGGAGCCTGGTAGGTGATGCCCTTTTACACGTGACGTCACCCCGAAATCCCCATCCGCTGTCCCGTTTTCTCTTAATGGAGGGTGCTTTTTCTTTCAAACAATGCAATGT

GAGGAAAGCAGTTTTATGCCGCTTTTGGATTACGGATTTCTAACTACTTACTAACATATTGGGTCCTTACCTATGAAATTGGTGTTTTTTTTCTCTCTTAGTATTTTGTCTGTAACTATTAAGATTCAGAGTATTCCTAAAAAATAATTACCTCGTCTAAAAATAGACTATATTTTTGAGCTCTTTTGATTTGTTTTGATAATTTGTTGTAAGTTAATTGTGTTAAAATTCGACACGGAGACAACTCTTTTTAAGTGCCTCCCGAATA

CATCATTTTATTTGAATTCACTTAAAAACGATAAATAATTTTTATGAAGTGTGATATTACACATATTACTAGAGCTTGTCTAATGATACATGCCAAATATTATTATCTTTGGATTTGTCGTTGTGAAGAAAACTGAAAAAACTTTAGCATGGAAAATGCAGAATTCCCCAAAATCTCCATACAAAACACCAATTTCATAGGTAAGGACCTAATATTAATACCGCATATTAATTACTAACACCCTTACTAATAAACAAGGTATAAGGTT

GAAATAGTTATTCAGTGTTTTGTCTTCGTCAGACATTCAGAATACAGTGGTTGTCAGAAAGTGACAAAACACTGAATAACTATTCCTTCCTTATAACTCGTTTATTAGTAACGGGGTTAGAATATTTAGAATGTGTCCCAATACCTAACTTTTCATCACACAAACATTTGCCTCGACTTGGCTTTGAACCCAGAATCGCTAGAATCGCTTCCATTCTTTCACGGAGGTCGATTCCCAATAAACTCAAAACCGAACGTTGAGATTACCA

TTTTCAAAGAATGGAAGGGTTTTTGTTGATAAAGTTGAAGGTTTTCATTCATTTTTTAAATAAACGTTTATTTTCAATAAAACTAACGCATAAATAAGTGGTCATGAAGTAGAACATTTTGCGGCACAGCTTCAGTACTACGAATTATGCATGTTCTGAAAACATCTTATACAATAGGTTTTTTTTGTAATAGAAGAATGTCTATACTCACTGCCAAAAAAACAACACATGATAAGTATTACTAGTACTAGACCAATGAAAATAAATT

CCCCAATATGGAAATATGTTTCCATATTTTGAAGTGGCTCTCCGAGATTCCTTTGTGTCGTCAATAGAGTGCTCATGTTAGATTACTGAATGGTCTGTAATAACGCGTATAGAAAAGTTTTGTTTGTTGTTCCAGGATGGGCTAGCGCCCCACTCTCACTATATGGTTATGATAAGCTATATGTATGTTTACGATATGTAGTTATTTATACGTCGGCCGTAACGTTACTAGCCAGATCTTTGTGCATGTTATGTTGTTGATTGTGACT

GAGTAGGTTAAAAAAATTTTTGTTATGGTTTTTCAAAATTTCTTATAGCAGAATGTCAAATACACTATTGGATGCTACTATGGCTCTACCTTTAACTATTTTGTATGTATACATATGTATTCATATTTCCTATTATAATTATATTTCTTTTCTTGTTTTTTTACATAATTTTTACGTTGTCAATTATAGCTGTTAGGTATCGGAGATAAATAAGAAGCCGAGCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTC

AAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAGAACTACCCCCCAGTGAACGCACGAATTCAGGGTGGCGTACAGGTACTGGCCAAGGACACCAGAAGCCGGGTCGCCCGCTTAAACGACCCCGCTCCAGTGGCAACATCGACGACTCTAACTATGCCATCTCGCCCGGGGTACAGTTTTGTGATCCGGCCGCGAGG

CCACGAACCACGGGGGAGGGTCCCATCAACGATTAGAACCAGATCTCCAACTTTAAAGTGCCGGCTTTGCCCAGGCTTCCGCGGGTAGAGTGTAGGCAAGAACTCCTTAGTCCACCTCTGCCAAAAGTGGTCGGCGAGACGGAGGCCCTTTTTGAAGGATAGCCGACCAAATAAATCAGAGTCAGAGAAAGTTGAGAGAGGTAAATCATTGACGGGACCCAAGAGAATGAAATGAAAAGGCGTGAGCGCCTCCGGCTCTCCTGGCTCC

ACGGAAACATGAGTTAGTGGCCGGCTGTTCACAATGTTTTCCACCTCCAGAAGCAGCGTGTGCAGGACCTCTTCTCTGGGAGAGCGCTCCTTCAGGGTCACAGCCAACGAGGTCTTGACGGTTCTCACAAGCCGCTCCCAGGATCCTCCCATAAATGGGGCTGCAGGAGGGATGAAGACCCACCTGATTTTCCTGTCAGCGCTGAACTCGTAAATAGCAGGTAACAAGCGCGCAGCGCCGACGAAAGCTGTTCCATTGTCTGAGTAGA

TGGACTCCGGGCACCCTCGACGAGCGATAAACCTGCGAATCGCCATAATCGCAGAGTCTGAGGAAAGCGACTCTACCACTTCGAGATGTACTGCTCGGACTGTCAGGCAGGTAAACAACGCTACATAGCGTTTCAAATGGCGCCTTCCTTGCGCAATCGTGACTGGCCCGAAGTAGTCCACTCCAACATGAGAAAACGGCCTCGATTTATAAGCTAGGCGTGCCGGTGGTAGGTTTCCGAAGGGTGGTGTCACTGCTCGGGCTTTCCG

AATACGGCAAAGATTGCATCTGGATAAAACAAGCTTCACCGATGGTCGCAGCCGAAGAATGAAAAACCTTACTCGTAGCGCGTTGACCACAGTCTCAACTCCGCTATGCGCCATCTTTCGGTGGTAGTCTCCGATCAGCAATTGCACTATTGAATGTCGACCGTCCAAGACAATTGGCTCCTGCTCATGGCCTGGTGGCAAAACTTCTGTTAGGGAGACCCTGATAGAGATCCGGAGGAGATGATCAGACCTAACAATGAGTGATACA

TTTTTAAGCCTACTGTACCTGGGGAGTGGCATAGAATTTCTAACGCATGTCATTTCCTCTGCCAAACTATCAACTTGAGACTTAAGCAACAATTGGCTTTCGGCTCTAATTACATTATCTGCCGAAAGTAAACCCGGCATTTGGGATGGACGCTTTAAGAACACGTTCGCGCAAAAATAAATACGGGCAGTTGCACGTAAAAGGCGTGTCCACGAGCTGAATTTCGTAAAATCTGCAACTACGGGTACAATAGAAATTAGTACTTGAC

TTTCAGACGAACAATGAACCATTTCAGGCAAAGGAAATGACACTTGGGGTTCCTTGGGCCAATCAGAATTAGAAGCCAGGAATGATGGCCCTAAGAACCAGCGGGAAATTTTGAATTTGTTGTCTGGTCTCGTAGCGTCGTCCGCCACATTTTGTGCTGAAGTCACATAATGCCAATCCGAGACATTAGTTATTTCAGTAATTTCCCCAACTCTAAGAGCCACAAAGGCTTTCAGTGAGCGGGCGTCGCTACGTATCCATCTTAAAAC

GGTCAAACTATCAGTCCAAAAGTAAATTTTGAAGGTTTTCGTCGGTGACAGTTTAGAATGTAGGCACCGAGACGAGCAGCGATTAACGCAGCTTGCAATTCTAACCGCGGAATTGTCGTTAGTTTGAGCGGTGCTGAGCGAGTCTTTCCGGCGATCAACGATATCTTAAAAGTCTGATCTGAATACACAAAACGCCAGTATGCCACACACGAATAGGCAAGCTCGCTTGCATCCGCAAAAACGTGGAGCTCAACCTCTGAAAAGGCAG

AGTCGCTGAAATAGCAGCGAGGTATATTGACATGTGCAACATCTTCAAGGTCTTTAAACCAAACGTACCACAGGGCTGATAGACTTGAAGGCAGCTCCGCGTCCCAATCAGAGACGGCCTTCCAAGTCTTTTGAAAAAGTATTTTACCCTTGACTGTAATAGGACTTAGGAGACCTAGAGGATCATAGACGCTCATCAACCTTGACAATACATATCTCTTTGTTAGGATGCGAGGGACAGTTACAGTTAAATTTCGCGTAGGGTGAAT

AGTATCAAGTGTTGTATTCCAAGAGACTCCTAAAACCTTAACGCTGTCATCTGAACGCGTGGCACGCAACTCTCCCGGAACCAATGACAAAGCGCCCTCTACGTTAGAAACCCAGGACCTCATCTCAAAACCCGCACGAGAATGAACGTCAACGACATCCCTAGCCAGATGAGCCGCCTCAGAAACATCATTAACCGACGTTACGAGATCATCCATATAATGGTCCTGAATGATTACATTAGCGGCTTTCGGAAACCTGTCCTTATGC

TGCTCAGCATTCAGGTTCATTAGATAGAGAGCCGTGAAAGGACTCGAGGTAGCGCCAAAAATCATGGACGACATTCGGTAATGACATAAGGGCTGAGACGGATCAGCGCGCCACAGAAACCGCTGAGCGTCACGATCTAACATGCGGATTTTTATCTGAGGGTACATCTCCCTAATATCGGCGTTTAGGGCTATCAGTCCTTCTCGAAAACGCATAAGGACCTCAAGAAGGGGTTGAAGTAGGTCGGGCCCTGACAACAAAAGGGAGT

TTAAGCTTACTCCTTGAGCCTTTGCTGCGCAATCGTGAACAACCCTAAGCTTTTTCTTCTGTCTATGGACAACACCAAAATGTGGCAAATACCATTTTACCGGAGTATCAGCGTGTGGCATCTCGCACTTCTCAGCATAACCCTTGTCTATCATGGCTTGAATATTACTACGATACTGAGTTGCATACTCTGGGTCCTTAGCCATCTTTTGTTCTAGAGAAGTAAATCTGCGACGTGCTAGAGCGTAACTATTAGGAAGCTGTCCCAA

ATTAACATCTGACCGCCATAGTAGCCCTGTCTCAAATCGACCAGAAGGGAGTCTGCGTGTGTTGACACGAAGGGTTTCTATGGCGAAAATATCATCCTTGTCAAGTCGCTCTCTCCTATCAATACCAATTGACTCTAGTTTAAAATTTTCTCGAATAAGATTCTCTAGAGAATTATCTCGTACGTGATTGACAGTAGAGTCTGACCGGAAAACTCTACTACAGACGTTCTACTTGTCGGGAAACCGTAAACTACCCATCCTAAAGGAG

TACGACATGCCACAGGCTGAGCGCGTGAGATGCGCCTAACTTCCGACGCTACCAGCAGGTGCCAATTGTCCATGCCGACTAGAATTGATGGTTCCACGTCCGAGTAACTGCACAAATTAAGACCGTTAAAATGCTCCAAATTAGTATCGCATCTCGATAAAGACTGACGAGAGAGCCCAAGATCGCGAACGGAACGCACATGTTTCAAAACATGTGGTTGACAAGTCTTATCATGACCTTTAATTGTAAAGCTAACATACTCCACGTC

TACTCGCTTTTCTAGATTACGCACACATGAGATGATAACACTCTTTTTTAGCCCCGTAACACCGATAGCAGACGAAACCTTAGCGTCTATGAACGTAGCAGTTGAACCCTCATCCAGAAGGGCAAAGGAATCTATGCTGCCACTCGGACCACTGACGGTTACCGGCAAAATCTTAAGAAGTGGTCTGGAATCTTGCTGCTTACATTGATCGCCATCTGTTTCCAAAACTGTCTGCCCCAACGAGTAAACCTGGGTTGAGGAGTCGACT

TTAGACCTATCGTGCAACGCATCGAGTTCGGTAGTCGGTTCTGCAGAAAATTGAGAACCTACAACAGGATCGACATTGGTAGCTGCTTCATGCAAGAGAGAATGGTGGTGACGTTTACGTCGGCAACCGACGACCTTACAACATTTCTTGCAGTTTCTATAGCTGTGACCACCTTTCCTCAAGCATTTGAAGCAAATAAAATTGTCTCTTACCCAACTCAATCGTTTTTCTGCAACCAAATCACGAAACATCTGACACTTCTGGATTT

CATGCGTAGCCTTACAATAAGCACAACCAGGTTCGCTAGAGGTGTTATCTGTGTGACTTGGAAACGATGACTTAGGAACTTTACCTGGTTGTTCGAAGCCTTCTTTGTGTCTCTTGACAGAGCGAAGATGTCCATAATTAGAGACAGGCCTTCCAGTCAAAGTCGTTTGACAGACTGTAGAGGGGTGCTGGTCAAGAGTTGCTGAAGTGAGGGAGGAGTCGACGAAGTCAGCCTGGACAAAATGAGCATGAGACCTATCTGACTTAGG

TTTAAAATCTGTCAGGATACCATATCTTAAGTGCAATAAAGCCTCATGCGATAAGAAATCAGAAAGCTTTTCCAAGCGAAACTTATTTTCATGCTCTTTCTGAAAGGCAAACGCCAACCACTTCTCCCTCAGGAGAGGAGTAAGCTTTCCAAGATAACGTTGCACAGTTCAGGGTTAGCTAAATGGACTTGCTGCTGCAGAAGCTGAACGGTACTTATGCAGTTGCGTACCTTGCATGCAAAAACATTTAGCTCCTGTGCATCGTACC

CGCTTAACAGCGTAGCCGCTTCTGCGCAAGAACGGAAGAACATTTGGTGGCGACTGAATTACCGCTTGAGGAACTCACCTGCGCCCTTTGCTTCGAACGTGACGTGGATGGGGACGGCGGCGGGCCCAACTCTGCGACCGCCGCGGCGGCATTATCCGTAGCGGAGTTAGCCTGCTGCTTTGCGATAGCACGGGTGATCATGCCGCGAACTTTTAAATAACTTCTGACACCAGAAACTGTTAGGTATCGGAGATAAATAAGAAGCCGA

GCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTCAAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAATAGCTATTAATTTTTTTGTTGAATAAAATACTACACATCTACTCTTACCGCGAATCGGACCCTCAGATATGGTCACGAAGGTCGTAAATATTTTTAAGACGTTGTTTCAAACCATCG

CGTTTGAATATAACGTATCATCGTTTGACACCCTTCGGAGAAAAATATTTGCCCAGCAGTGGGAGTCTTTCGACTGAGATGTATACCATGGCTGAAAAAAAGATACTGAATAAGAACACTCGAAAAAAAAAATCGGCATTCGGCGATCTTAACGCTATGCAGCAGTCTCTTTCAGAAAAACTGCATCGTAATAATACTCGTATAATAGAAAAAATCAATCGTTACACAGTTATTTTCAAGAACATTTGTATCATATATTTGTAAATAT

TCTTTTTTATATTGCAAGATCATTTCTTAGTTATTGTTAAATGGTTGTTCCTTTGCGACCCATTCAATGTCTGCGGTCACCAGTCGACCGTTGTTGACAGCGTGTCAGACTGAGAAGGGTCTATCGTTGGAATAACTGACATATTAACACAGTACTTGTACACATGGAAACGATTTTATATAATACGACTAGAATTCTATTGTGATAGTTTTTTTGTTGATGCTAATGAATACCTGGCAGTATCTATAGACGTAGTTACAATTAACCG

TATTTTAACAGAATTAAATTCAAATAAATAAGTGTGCCTATTAGTATTTTTATTAGTTACTAGTGGCTTACCGCGGTTTCACCCGCGCGGTACCCGTTCCAATGGGAATATCGGGATAAAAAGTAGCCTGTATTCTGTATACCAAATTTCAATTAAATTGGTTCATAGTTTTGACGTGAAAGCTAGACTGACAGATAGAGAAATATACAGACAGAGTTACTTTCGCATTTATTATTAAGTATGGATTGTTATTATTTTATTTTAATTT

ATTTACTTGGAAGAATCGTTCACTCAGGTTGAACGTAGAACCCTAATGGACCATCGATTTGTAAAATAATAATAATAATAATATCCTCAGACATTCACACCGATCAACTAGCCCCATTTTAAGCAACTAATGCTGCTTGTGTTACGGACACTAGATGACGGATAAATATATTTAATAGATAATAATAATAATAATAATAATAATAATTTATTTCGATAACTTAGTCTACAATTACAAATTAAATGTGGGATCTCCCTTTTAGGCAAGA

CATCGCCTATAGATAAATACATATAAATAACCAGAATATATAGAAATATATATAAACATCCAAGACTGGAGAACAAATGCCTGAAAATTCATCACCCAAACATTTGCCCCCACTGGGGATCGAACCCAGAACCTCTCGAGATGGCGACGCCTTGAGACCGGCATACAGACCACTCGACCACGGAGGTCGTCAAAAGAACGAGGAAATGCGTTGTATGCGGGCTGCACAGGACCCACCTGCTTAGAGAATTGGAGATCCTTATGGGAGG

CTTGTATTGTATCCAACAGTAGGTGTCTGTCACCTGATATGACATATTGGCGAGTTTAGGTCATTACCTTGGTTTTACATACAAATAACGTATACTCTAAGGTTCCTGAATCTATATAATTTAATTATGAAAATTATCGCATTCGTCTCTGGAATAACCTTCCATTACCAATCAAACAATTCCAATCCTTAGCATCCTTTAATGATCTGGTCCATACCCATTTCCTTCATCTTTGACTCCTTCCTCCAACACACATTGCAGTTTTCGC

CTTTTCTTTGTTGCTTTTTGTGTAAGAATATAGATAATAAGAATATATTGCTAAATAAAATATTTATACAGGATATTGTAGCATTTATAATATAGTATGTATTTTATGTATGTATATAATATATTATGTAATTTATATGTAGTAGTTTATATTTGCACCTTATATATGTTACCTACCTTTTCTTTCATTCTATCTCTGTCCAAAGGTTGTCTGGAAGATATTGCTATTAGCTATAAGGCCGCCTGTGTTGTATTGTATTTTTTTACTA

TATTTCTGTTTTAATTAGTTGCAATAAAGAATTTTATCTATCTATCTATTGCCGATAACTTTCATGAAGAATTTTCCTATTCATGAATACCATGAACAACAAACCTAAACTCACCCTTCTCACTCAAGTTAACTTATGCCAACTATAAATAATAAAAATAAACGAAACCCACATTATGATTGTGTAGTAAAATCTGACAGTAAATTAATATTTCTATTTGGTAAGGCCGGTACATTTAAAGCGTTTTGTTTTAAAAATACAGCTGGCT

GTGTGCCAAGCAAGCGAATACTATATTGGGCCAGCCTGGCTAATTGTTACTTCTGAATGCAACGAGGGAAATTTCTACAGCCTCCGACAACATTGTGGATAATTTTTCGACTGTATCCAACGTTGTAGTTCCTTTACAAGAATACAAAAAGCTCTTTAAACCTATTATGAGTCCTGTGCGCATTTCGGAATTTCGAGTTGCGTTCGAGTTTTTCACTTGTTTTACCAGGAAGTTATTGTAGGGATGACTGTAATTATGATACAAACAT

TTTTGATGATGTTGTAAAAAAAAAGCTATTGCATAGCATTTTGTATCAACTTATGCAATTATAATTTTATTTATTTATTTTATTCATGCAGTATTTCTTTTTCTTTTTTATTATTTTACAATACATGTGGGTTTTATTATCTTAACGCCGTGTCGAAGACTGCCGAACTGAAACGACGTTAGACGATGCACGGCCTTCAAGCCACACCTCCTTGCGCATCGGAGTGGGGAGCGTGAGGTTTTTTCGTTACGGAATTTCTGGATTCGGT

CCCCGCGCTCAAGGCCCGAGATAGAAGCTATGCAATAGCTTAAAAGCTTGTAGAGAAAGAGATAAAACGCCTAAAAATGGAGACCTAACATATCAGCAAAATATGGGTGATAACACCTCCCTACCCCTTTGCAGAAAGAGGCGCGATGATATGTTATTCCATATTTTTTTTACATATTTACATGCAGTGTTATTGTAAATGGACAACCTCCGTAGCCGAAATTTGTACGCTGGTTCGAAGGAATCTCGTCATTTTTAGAGGTAGGCTC

ATTTGATCTTCGGTGATTTATTCTCTAAAACTTATATGTATTGAATATTATGGGCCCTGGCAGGGAAAGTACCTTACATATTTAAAATAGATAAATTTACATAAAGGACTAACTTTTGTTTTTAATAATTAACATAACTGATCTCTCAGTTTTTTTTTAAAAAAGCTTACCCAAACCTTCCCTGGAATATAAAATAATTGTTAGACATGTTTAAAACGTAAAGCTTAATAAAGAAATTAAATTTTCAACAATGACTTAATCGGTTAGT

GGAATTAAATCGCGAACTTTTTTTATTCATTTTTTCCTTATTCGATTCTCGGTTTGAGTAAGCTAGCTAGCTATTTTGAGAATATTTAGAACTTAGTTATGTTTCTTATTCAAAGTAGAAGTATGCTCTTGTGTAAATTATATTTTTTTTTAATATTAGCTAATAGCTCCAGGGCCCACAAAATTTTCCACTCTGTATGTAGGTATATACTTATCTATTACGCTCATTAAAACTTATCCGTCATTTAGTATCCATAACACAAGCAGTA

TAAGCTGCTTATTTTAGGCTACTTGATAGGTGAAAAAAATATGTATTACATTCATACATACATTCACGCCTCTTTCCCAGGGGGGTTGGCAGAGATTACAGATCTGCACTTGCTACGATCCTGCATCCCGCACATGACACACACTGTCTCTACCCTCATTACTAACTTTATACATGCCCTTCGGTTAATTTTAATAATTATTTATTAGAATATTAAGCTTTATTATGATTAATAGCGATGGCAATTCGTTAGCTTATAGGCGCCATTT

TCTTTATCCTCTACTTAAGTAGTAGCCAGCAATTTTAGGGCAGTGACCTAAGGTCAACGACTTGTAAAGGATAGTTCTTATGTTTTAAGAATACCGCATAGTAGATACGAATAACGTTGCAGTCTAACCGTGCAGAAGATTTTTGTCTTTCCGGATGATTAGATGTTATGAATATGAGGGAAAATATGCTTTAAGTATGAAGACTATAAGGTCTAGTATTTAATTTGATGTTATAGTTATCTTCGCACTGCTCTATAGATTCTTTTAT

ATAAAAATGACTTTTATTTTTTTATTAACAGCAACATTCTAATCTAAAATAATATAGACCAATTTTATATATTTACTTTTATATTTACCTGTTAGATTGAGACTGAGAAATATCAGTCGAATGGGAGTGTTTAAAACGCCTGTCTACCCCTTTGGGATAGAGGCGTACGTTTATGGTTTATTTTGATGTTTGTTGTATGACATATTACACTAAATCTCTACAAAGAAGAACAAGACGAGCCGAGTTGCCATGCATCGGGCACAAAGTG

AAGCAAGCAATATTAACAATGAACTGTACATAACTTTTAATTCAAACTCGGTAGAACTCGCGAATCCCGCGGCCTCTCCAAGGAGTAGTGCCCTCTGTCCCAATTCAGCTAAGGAATAGTCAACGTAATTACTAAAGCAGACTTATTGTCTCCACCACATGTAAACCGTTTGAATCATGCATTAATAAAACTTCATGTCCCCAACAGTGGGCACTGGCTTTTACCTGACTTCATATCTTACACATTTATATCCTTAGAGGAGTATACC

GAGGGGTCATGATTTTTGTAATCTAACAATGTATGTTTGTATTTTAAATTGATAATCCAGACTTATTATAAATGCGAAAGTAACTCTGTCTGTCTGTCTCGCTTTCACGTCAAAACTACTGAACCAATTTAACCGAAATTTAGTACACAGATAGTCTAGAACCTGAGAAAGGACATAGGCTACCATTTATCCCGATATTACCATGGGAACGGGTGCCATAGGGGTAAATGTAAAAACATTTAAATAACGCCTCCCAGAAAAATGAAGG

ACAAATTTACGCCACTATGAACTTTGCTTCTAATAATAGAGTAGGAGTAAATTTTCAATTCCACCAATTGAGGCTACGGTTCTTATTTTAAAAAATCTTTCAGTGTTCAATAGCCTATTTTTTGAGGAAGGCTATAGGCTACATACCATTAATCACACTATCTCAAAGTCACTATTCTACGCGGATGAAGTTGCGGGCAAAAGCTAGTTGATAAATTCATTGATGTATTGATAATTCATAGACCATCAAGTCATTTAAAAACGTAATA

AAAAAAAATGTGTTAAAAGCCTCAAAAAAAATTATATAATTTTTTATGCTGGTGGCTAACAGAAATGAGTAATTTGGGGCCCAATAAAAAAAGGGTCAACTAGCCTAATATTTTAATTTCTTTTTTGTACTCATTATATTAGGTCCTTTCCTATGAAATTGGTGTTTTGTATGGAGATTTTGGGGATGTCTGCATTTTAAATACTAACATTTTATCAGTTTTCTTCACAACGACTAATCCAGAGATAATAATATTTGGCATGTATTAT

AAGACAAGCTTTAGCAATATGTGTGTGAACACGCTTGATAAAAATTATTCATCGTTTTTATGTTAATGCAAATCAAATGATGTATTCGGGAGGCAGTTAACGTTTAAACATCAGCCTATGTTTGTCCACTGCTGGACATAGGCTTACCCTAATGCATCGAGAGGTTATTCGAAAGATTAATTGCTGTTTAGCTGAAAGGTAAATTGTGAAATCAATCTAGTTTCAAGAAATTAACTGTTTATCCTATGTGGAGTTATCAGTAAAATGT

TTTTCCACTAAATTGTTAGTTAAAATTTTACACAGCAATCCAACATAACTCTTCTAGTCATAGCAGCAGAGTGGTTTTTTAAAAACCGACCGTTTGTTGCCGACACCACACCTGTTTCGCAATTTTTACAACATTCAAAACACCACAAACTCATTTAAACTACACTACATCAAATTGTCACGTTAAATTTATTACAATAGAGTTGCTTTTTGTTACAAAAACCTTTAAAAATGTATAGTTTTACGATATTGAACTTTAAAGCGGTAAT

AATAAAGCCTTAATATTGTGTTTTAAGACTATTATCTAACGACGCCGTTCGCGTAATTTTAGTTACATTGATCCAATTAATATGAAAACTTATAGCTGCTATCGTGTTTCGACACTAGATTTTCTTTGACACCAAAAAAGTACTAAGCTTATTACATAAAAGGCCAAATAGTGTTATAATTGTTTGTTTTGACAATCACTGATGAAACTTTGATATATTTTTCCCGATGTAGGTATTCGTCTTAGTACACTACTTAAATAAATAGAAC

ATTTTACTAAAATGTTCTTCATTGTAGTAATTATCGCTATTTCTTTTGAATCAAAAATTTAATAATATTAACGCGACGTTTCCCGATCACGCTATATTCCCGAATGGGTAGTTTTAATCTGAGCAACTTTACAATAATAACTTTTTAAATCATAGTTGATTAATAAATAAATTATTCAAAAAAAAATACAAATTTAAGACTAAAAATACTTAAGACAAAATTATACTTACAGACTTTTTATTATCATTT

19,820 BP HIFI READ, PREDICTED QV: 3319,812 bp correct, 8 errors

99.96% accurate (QV34)

>m64089_191020_002935/346/ccsGAGTCAACCGCTCTACGCACTGACTCTCGTAAGAATGAGCTTCGAGAACAAAACAAATAAAAAGAGACAGACAGCAAATTTAAAATCGGAAACCAGGAGAAAAAATGGCCAGAGAAGAGCGCAATGACAGCAACGAGGCGCTAATATAGCTATGGCGCCTCGTTACAACGTGAACTCTCCGAAATCGTCTGTTGTTCTTTTTAAAATAGTTGTTATTACATAAATAAGCGGTCGATGCCTAATACCCATTGATTTTGTAGTAAATACA

TATAATACTCGTATTATGAGTATTTACTACACAATATTTTCAACGACATGGCCGTCATTTTAATTCCGTGTCTTGGATTTATGACGTCATAAATATGATGAAAAGATATCGCTGGGTATTCCATGATTAGATTTTATGTGCGCTTTTATGATCCAAGACTCCAATAACTTCGAAGAATTCTTAGTAGAATCTAGTTATTATGAGATTAAAATAAAACTAGATAAACAACTAAGTTGTTTGAAATAATTAATTTTATTCAGCAATTTTT

GGCTTTCCAATTCAATTAGGTGCAGCCTATTAAAAACTAAGTTGCAGTACGCCACCACTTTTAGTTTTTCACAGTCTATTCCTATTATAAAGAACAAGCTGTGAATAAATTACAGTAGTTTCCCTTTCCTAAACTGAAAATACACACAATTATAATCTTATGAAAAACATAAATTCGTTTCATAAAGCTTATCGCTTTGTTTCTTTTTAGAAAATTCTCGGGCTACTTAGACCTCGGCGATGTACTCAGTACCTACCCAAATTGCTGG

ACTGGACTGCGCGATGCAATGTTCTGCGCATGTCCGTTTATATATAGGTAGCCTATTGATATTTTGTTATGACTTTCGTATGTCCTGGAGTAGCGTCCCTAGCTACACCGGCTTTTGCAGTTTTATGCACTCCTGGTAGTATATTCCAAAAAACATGTGACTGCGCCGTGCGTCAGATCGTTATGAAAGCTGATAAGCGACTGAGCGAATATGTCATTATTAAAGAAACTCCGCCATTTTGCTGCCGCCTGCTAACACTATGTGCAAT

GTGCATTCTTCTTCGATTTTCCGATTTTCATTTATGAGTGTAAAATTTTTATTTTGCGATGTAATGAAACTTCTTAATTAATTGAAATTAAACATATGTATAAAGCTGGGGAAGCTCCGTGGCGTCGTGGTTTGAACGATTTGGTTTTGATGTTAAAAAAAGCTGTTTATTTTTACATATTTTTTTATGGTATGGTGAACAATGAAGAGTTATCTATCCATCTATCTTACTTTCCCCGTGTTTAGACTTCGGAGGAACCGGTGCAACG

ATATTTACAATCATTGAAACACATTTAATACAAAAAAAAGTTCTTAAAAGAGACATTTCCTATAGGACATTTCCTGAAATATTTTGTGTAACACTAATGTTTTTATTATGCAACACAATACCTTAGCTACATATTATAAAAAAACTCACAGATATTGTATATATTTGTCTTTTACCTTATATAACGCATCTGTTTGGATTTGTACTGAAGCCATGCGTGCCAACTTTTTAGCATCAACGGTGACATGACAGCCCTTGACAGCCGTTTT

TAACGATGAAATAAAGGAAAGAAAAATTTTAATTTTATTGTGTTATGAGTTGTTATGGGCTTAAAAAACATATTATTTTTAATGGTGACGGTTAACAGCAATGAGTCATTTGGGACCCCATAAAAAAGGGTCAACTAGCCTATTAGAGTTTAATAAGATTAGTCTTGTGGTCGACAGTAATCCAAAAATACCAAAAATACAGTCCGCGTCCTAGGAATCTAGGAATTTATGTATTCAACATTAAAGGCGGATCACTACACGCCTCGAC

CCGTAAATGTGTAGCCACCGTAATGTTTGTATTTAAACACTTCGTCACATTAGAAAATAAAATAAAATAAATTGCCTATTTATTCGTATAAACAAACACATATTATTATATGTAAAATTTAAATAAAATTGCGATCATTAAAAGTTGTGTTATTGACTAGTTCCACGCAGACCAATGGTAGCGATGTTTTTCTCATATCTGCGCATGCCTGATGAGTAACAAATCAGAGGCCAATATTTACTGTTATTTATACGTTATGAATGGGAGC

GTCCGTAGCAACTGCACTTCCTTCAATACTGTGCATTCCTGGTAGTTGATGGACAAAGGCAACGTGGTTTGCACACTGGGTCACGCCCGAAGAACTATTTTATTATTTGTCCGCTGTTGAATTGGTCAGATTGTTTTATTTAGAACAAACGATCCTGTTGCCATGCACCGGGCAAAGCGTAAATTAATCAATTAGTTTTCAATTTTTTTTTCACAGGAAAAAAAATTAAAAACTATTGATTAATTACAATTTAATCTCTAACGACGTC

ATATTGATTTATTGATTCCATACTTTGTAAAACATTAAGTTAGTGTGTGTAACTTCTGCAAAGACAAAAAATAATAAATAAAAAATAAAATTCGAACTTCGAATGTAGATGGTGCACGTTTTTCTTTGGTTTCGGCCGGCCGGCGTCCGTAACAACAGAACCTACTTCTTGTACACTATGAGTCCCTGGTACCCCATCGTATAGAGAACGTGACTTAGACGGGGTTCCAAGTTTTCAACAATGTTTGCTTATTTGTTTATTTATTTAT

TTATTTGCATGTCACATAATCACTGTACAGAAATACAAAGGTCTTAAAAAATAGGATTGTATACATGTGACACCCTGCAAGGGTATAGCAATATATAATTATAATATTGGACCAGAGGGCCCTAGTTACTTAACAATTATATAAACTTATTTTTAAATTAAAATTTTATAAGCAACGTAAAATAAATTCTAATACTCGTATTACTAACAAAATTTAACAGAAACAAGAAAAACATGAGATCAAATTTTAAATTATTTACTTATATACG

TAACGTTATCTACCTTTTAATTACAAAAATTATTATATAGTGGAAGTTATTCGTTTTCGAAAAATTCCTTTAAACTGTAAAAACATTTCTCGATCAAGAACTTTTTTAAGTGCTCAGTAAAATTCGGGAGTTTCTCAAAGTTTTTGATGTCGTTTGGGATATGGTTATATATTTTTATAGACATAAAATATGGACTAGATGAGACTAATTTTAATTTTGTAAAAGGAATTTTAAGTTTATTTAAATATCTGTCATTTCTTTTGATTTT

ATTATTTGGTGAGAAAAATTCTTGATGTTTTCTGGCAAATTTACATGCTTCTAATATGTAAATGGATGTGAGTGTTAATATTCTGTGTTTGATAAAGTGAGGTCTGCATGATTCCATTTGTTCTATATTTGTCAATATTCTCAAACATCTCTTCTGCAATATAAATAATTTATCTATTTCAGAGGAGTTACCCCATAAAACGACGCCATAGGAAAGAATGGAGTATGCATAAGCATAGTATGCTGAGAGAGCTGTTTGAAAGTTTGTA

ACGCGTTTCAAGTGGTGTAGTGCATATATGAAAGATGATAATTTATGTTCTACTTTTTTAATATGCTGCTTCCAGTTTAAATGTGAATCTAGTTCAATGCCCAACAGGGTTGCTGTGTTCACTGTTTCTAATTTTATATTTTTATAACAATATTGTACCTGTAATGAACTTTTTTGATAGGGCTTAAATTGGATTAATTTGGTTTTTTTAAGGTTTAGTTGGAGATTATGTGTTTCTAGCCAGTTCGTAATGTTGTCTAAAATAAAGT

TTAATTTAGTTTTAAGTTCCGATGAGTTTGAGCAAGAGATAAGCACTGAGATGTCATCAGCAAACAATACACACAGATTATCAAAAATATCAGGTAGATTATTTATATAAATGAGGAATAAGACACATCCTAGAACGCTACCTTGAGGTATTGATCCGGTAACTTGGATGGCATTAGATCTGGTGTATGTGATCATTCCTGTCTCAAAGTCTGTATTTTGTATTTCAACGTATTTGGAATTTGGAATTATTTAGTAGTATTTGGAAGT

ATTTGGAATTGTTGAAACCAATAAATAAAACTTATAAAATCCTTATTCTACTTTCTACCATTTCGTGAAACAAACAGTAGATGGCCGATGTTTTTGTGTTCTAGGCCCACTTACAGGAAAATTAATGGAGATTACATAGTAGATATTATATCTAGACTAACCCCCTTACTAATAAAATAATAAAAATAACCGACATCCACGTGGGCGGAGCCACGGGCGACCGCAAGTTTCTTATAAATCTGTTGAAAATTTACGTCAAATGTGAAAA

TTCATGGTCTTTCTTTTATTTCTGATATTATGATGTTATTTAGTAAAGAATAATCCTCACGATGAAACCTTTCGATCGGTATGATAAAGCTACAGGTTAATTATTTTTCTCGTGTAACGGGTTCGATTCCCGGTTCAACAATGTAATTATTTAAATATCTATGAATGCAGTTTAAATAGTTTCTGAAATTGAAATAAAGTCTTCTTTCAGTAAAACGTTCTAACTGTAATATTTAAGGTACTTTCTGTGGTACTTTCCCTTCATGTGG

CATAGCCGTGGGACGGCCTGTATATTGCTGCGTAAATAAATATCTTGCTGCCCAATTAATTATTGTGCTGATCAAATAATAATGTTGCTGCCCAAATAATTATTATGCCGCCCAAATAATTATAATGCTGACAAAGTATTTGTTTGCTGCACAAATTATTTTTCGGGTTACCTTTTTGTTGCTGACCAAATAATTAAGAGACTGCCTAATTAATTACTTCCCGTTTAAAAAACCTCGGAGTAAACAGAACAAGTCCTACAAAATTGTA

TTGAACGTTCATTTTCGATTCACATTAAAGCAAAGGGGCGTTCCACTAAAAATAAAAGTGTCTACGTGATTAACTAGATGGCGCTGTCGTACACCTAAATCGTGCTATAATTTTCTCTCCTATTCTATACAATTTTCAGTTGTTGTATCATGTAAATACCCTGGTCATAAAGTATATTGAAAAAGACTATTTGTTTTATAATTTTTCACTAGATCCTCAATAAAACACCAATGGGTTGTTTAATATTAAAAATATTTACTAATAGTTG

TGAACATAAGAGCTTCCATTCCAGATTAACTACAAACATACTCTTGCAAATATGGGATCCTGGTAGCTCTCCGCAGTTTGTCAATGTGACAAAATGAGGACACTAGTTTGTAGTTTCATTGTTTCCTAGTTTATTAGGCCACGCCTAATCGTATGCCATGCCACTCCACGTACGCAGTCATTGATAACCGTCTGGTTTCGATAATGAAAACAAGGATACTATTATTTTGGATGGACCTCGGATGGATGACCAAAACTTTGTGATTTCG

ACTTCCACTGTGCTTTGGAAGCTGTTGATCTCGGTGTGTCCTGTAGGATGTCTGCTTTTTAATGTGTAATGTGAAATATAGAAACTTCGTTAGAAAACTACACAATAGAATATTGTTGACAAGAGGCTATAGAAACCTTTAAAAAGTCAAATTATTTTTAAGATAACCATAATAAGACTTTAAAAAGTTTGTACATATACGTTTCTATTCACCTGTGTCTACCCCTTCGGCGATAGAGGCGTGATTTTTATATGTTATGTTTATATTT

ATCCTTTCTATTCTACATCTCCGTATCTTCTAAAAATGAAAAGTAATTTGAATATTGAATTTTCTGATCATTGATATGAGTAAGGAGGCTTAAGGGTTTGCATAAAAAAATAATATGAATAAAGTATTATCAAAGTAAATAAACCAAGAAGTAAATCTGGCTTTTCTCTATAATTCCTTAGCTCCGTTTGATTTTCCTATTAAAATAATTAGCAGTGTAAATTGAAAGAAACTCGATAATTACATGCTACTGACAGGGTGGGAGATCG

TTAATCAAATCGCAAGAGAGGGAGCCGGAAGACGTATTTCTCTCGCTTCCACTCATCACTACCTCCATTAACTTTCTCTTTCACTCACAAAATTCGTCGCATGCAATAACAGATTCACATTTTTCACAAATCTCTAAGTTTTAGAGTTTTTTTACTTTAAATTTTCCACGTCTAGTTTTAAAGTGTGGTTTTGTATAACATGAAATAAATATGAAAGCACTAGAGATAGCCTTATGAAAAGAATATATTCGTTGCTGCAGTGAAAATT

GAACCATAACGTAATGGCTGTACATTGTATTTCGTTTGCGTGATAGGCGCAGTGTGCTTCGTAGTTAACTCGTGATGTTGCAGTGCAGTGTTCTATTCTATTGTGGTAAGGGTGCAATTTGCTTGTTCCTGCAATATGCGTGTCTTCTTGTGGGTTATAGGAGCCTGGTAGGTGATGCCCTTTTACACGTGACGTCACCCCGAAATCCCCATCCGCTGTCCCGTTTTCTCTTAATGGAGGGTGCTTTTTCTTTCAAACAATGCAATGT

GAGGAAAGCAGTTTTATGCCGCTTTTGGATTACGGATTTCTAACTACTTACTAACATATTGGGTCCTTACCTATGAAATTGGTGTTTTTTTTCTCTCTTAGTATTTTGTCTGTAACTATTAAGATTCAGAGTATTCCTAAAAAATAATTACCTCGTCTAAAAATAGACTATATTTTTGAGCTCTTTTGATTTGTTTTGATAATTTGTTGTAAGTTAATTGTGTTAAAATTCGACACGGAGACAACTCTTTTTAAGTGCCTCCCGAATA

CATCATTTTATTTGAATTCACTTAAAAACGATAAATAATTTTTATGAAGTGTGATATTACACATATTACTAGAGCTTGTCTAATGATACATGCCAAATATTATTATCTTTGGATTTGTCGTTGTGAAGAAAACTGAAAAAACTTTAGCATGGAAAATGCAGAATTCCCCAAAATCTCCATACAAAACACCAATTTCATAGGTAAGGACCTAATATTAATACCGCATATTAATTACTAACACCCTTACTAATAAACAAGGTATAAGGTT

GAAATAGTTATTCAGTGTTTTGTCTTCGTCAGACATTCAGAATACAGTGGTTGTCAGAAAGTGACAAAACACTGAATAACTATTCCTTCCTTATAACTCGTTTATTAGTAACGGGGTTAGAATATTTAGAATGTGTCCCAATACCTAACTTTTCATCACACAAACATTTGCCTCGACTTGGCTTTGAACCCAGAATCGCTAGAATCGCTTCCATTCTTTCACGGAGGTCGATTCCCAATAAACTCAAAACCGAACGTTGAGATTACCA

TTTTCAAAGAATGGAAGGGTTTTTGTTGATAAAGTTGAAGGTTTTCATTCATTTTTTAAATAAACGTTTATTTTCAATAAAACTAACGCATAAATAAGTGGTCATGAAGTAGAACATTTTGCGGCACAGCTTCAGTACTACGAATTATGCATGTTCTGAAAACATCTTATACAATAGGTTTTTTTTGTAATAGAAGAATGTCTATACTCACTGCCAAAAAAACAACACATGATAAGTATTACTAGTACTAGACCAATGAAAATAAATT

CCCCAATATGGAAATATGTTTCCATATTTTGAAGTGGCTCTCCGAGATTCCTTTGTGTCGTCAATAGAGTGCTCATGTTAGATTACTGAATGGTCTGTAATAACGCGTATAGAAAAGTTTTGTTTGTTGTTCCAGGATGGGCTAGCGCCCCACTCTCACTATATGGTTATGATAAGCTATATGTATGTTTACGATATGTAGTTATTTATACGTCGGCCGTAACGTTACTAGCCAGATCTTTGTGCATGTTATGTTGTTGATTGTGACT

GAGTAGGTTAAAAAAATTTTTGTTATGGTTTTTCAAAATTTCTTATAGCAGAATGTCAAATACACTATTGGATGCTACTATGGCTCTACCTTTAACTATTTTGTATGTATACATATGTATTCATATTTCCTATTATAATTATATTTCTTTTCTTGTTTTTTTACATAATTTTTACGTTGTCAATTATAGCTGTTAGGTATCGGAGATAAATAAGAAGCCGAGCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTC

AAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAGAACTACCCCCCAGTGAACGCACGAATTCAGGGTGGCGTACAGGTACTGGCCAAGGACACCAGAAGCCGGGTCGCCCGCTTAAACGACCCCGCTCCAGTGGCAACATCGACGACTCTAACTATGCCATCTCGCCCGGGGTACAGTTTTGTGATCCGGCCGCGAGG

CCACGAACCACGGGGGAGGGTCCCATCAACGATTAGAACCAGATCTCCAACTTTAAAGTGCCGGCTTTGCCCAGGCTTCCGCGGGTAGAGTGTAGGCAAGAACTCCTTAGTCCACCTCTGCCAAAAGTGGTCGGCGAGACGGAGGCCCTTTTTGAAGGATAGCCGACCAAATAAATCAGAGTCAGAGAAAGTTGAGAGAGGTAAATCATTGACGGGACCCAAGAGAATGAAATGAAAAGGCGTGAGCGCCTCCGGCTCTCCTGGCTCC

ACGGAAACATGAGTTAGTGGCCGGCTGTTCACAATGTTTTCCACCTCCAGAAGCAGCGTGTGCAGGACCTCTTCTCTGGGAGAGCGCTCCTTCAGGGTCACAGCCAACGAGGTCTTGACGGTTCTCACAAGCCGCTCCCAGGATCCTCCCATAAATGGGGCTGCAGGAGGGATGAAGACCCACCTGATTTTCCTGTCAGCGCTGAACTCGTAAATAGCAGGTAACAAGCGCGCAGCGCCGACGAAAGCTGTTCCATTGTCTGAGTAGA

TGGACTCCGGGCACCCTCGACGAGCGATAAACCTGCGAATCGCCATAATCGCAGAGTCTGAGGAAAGCGACTCTACCACTTCGAGATGTACTGCTCGGACTGTCAGGCAGGTAAACAACGCTACATAGCGTTTCAAATGGCGCCTTCCTTGCGCAATCGTGACTGGCCCGAAGTAGTCCACTCCAACATGAGAAAACGGCCTCGATTTATAAGCTAGGCGTGCCGGTGGTAGGTTTCCGAAGGGTGGTGTCACTGCTCGGGCTTTCCG

AATACGGCAAAGATTGCATCTGGATAAAACAAGCTTCACCGATGGTCGCAGCCGAAGAATGAAAAACCTTACTCGTAGCGCGTTGACCACAGTCTCAACTCCGCTATGCGCCATCTTTCGGTGGTAGTCTCCGATCAGCAATTGCACTATTGAATGTCGACCGTCCAAGACAATTGGCTCCTGCTCATGGCCTGGTGGCAAAACTTCTGTTAGGGAGACCCTGATAGAGATCCGGAGGAGATGATCAGACCTAACAATGAGTGATACA

TTTTTAAGCCTACTGTACCTGGGGAGTGGCATAGAATTTCTAACGCATGTCATTTCCTCTGCCAAACTATCAACTTGAGACTTAAGCAACAATTGGCTTTCGGCTCTAATTACATTATCTGCCGAAAGTAAACCCGGCATTTGGGATGGACGCTTTAAGAACACGTTCGCGCAAAAATAAATACGGGCAGTTGCACGTAAAAGGCGTGTCCACGAGCTGAATTTCGTAAAATCTGCAACTACGGGTACAATAGAAATTAGTACTTGAC

TTTCAGACGAACAATGAACCATTTCAGGCAAAGGAAATGACACTTGGGGTTCCTTGGGCCAATCAGAATTAGAAGCCAGGAATGATGGCCCTAAGAACCAGCGGGAAATTTTGAATTTGTTGTCTGGTCTCGTAGCGTCGTCCGCCACATTTTGTGCTGAAGTCACATAATGCCAATCCGAGACATTAGTTATTTCAGTAATTTCCCCAACTCTAAGAGCCACAAAGGCTTTCAGTGAGCGGGCGTCGCTACGTATCCATCTTAAAAC

GGTCAAACTATCAGTCCAAAAGTAAATTTTGAAGGTTTTCGTCGGTGACAGTTTAGAATGTAGGCACCGAGACGAGCAGCGATTAACGCAGCTTGCAATTCTAACCGCGGAATTGTCGTTAGTTTGAGCGGTGCTGAGCGAGTCTTTCCGGCGATCAACGATATCTTAAAAGTCTGATCTGAATACACAAAACGCCAGTATGCCACACACGAATAGGCAAGCTCGCTTGCATCCGCAAAAACGTGGAGCTCAACCTCTGAAAAGGCAG

AGTCGCTGAAATAGCAGCGAGGTATATTGACATGTGCAACATCTTCAAGGTCTTTAAACCAAACGTACCACAGGGCTGATAGACTTGAAGGCAGCTCCGCGTCCCAATCAGAGACGGCCTTCCAAGTCTTTTGAAAAAGTATTTTACCCTTGACTGTAATAGGACTTAGGAGACCTAGAGGATCATAGACGCTCATCAACCTTGACAATACATATCTCTTTGTTAGGATGCGAGGGACAGTTACAGTTAAATTTCGCGTAGGGTGAAT

AGTATCAAGTGTTGTATTCCAAGAGACTCCTAAAACCTTAACGCTGTCATCTGAACGCGTGGCACGCAACTCTCCCGGAACCAATGACAAAGCGCCCTCTACGTTAGAAACCCAGGACCTCATCTCAAAACCCGCACGAGAATGAACGTCAACGACATCCCTAGCCAGATGAGCCGCCTCAGAAACATCATTAACCGACGTTACGAGATCATCCATATAATGGTCCTGAATGATTACATTAGCGGCTTTCGGAAACCTGTCCTTATGC

TGCTCAGCATTCAGGTTCATTAGATAGAGAGCCGTGAAAGGACTCGAGGTAGCGCCAAAAATCATGGACGACATTCGGTAATGACATAAGGGCTGAGACGGATCAGCGCGCCACAGAAACCGCTGAGCGTCACGATCTAACATGCGGATTTTTATCTGAGGGTACATCTCCCTAATATCGGCGTTTAGGGCTATCAGTCCTTCTCGAAAACGCATAAGGACCTCAAGAAGGGGTTGAAGTAGGTCGGGCCCTGACAACAAAAGGGAGT

TTAAGCTTACTCCTTGAGCCTTTGCTGCGCAATCGTGAACAACCCTAAGCTTTTTCTTCTGTCTATGGACAACACCAAAATGTGGCAAATACCATTTTACCGGAGTATCAGCGTGTGGCATCTCGCACTTCTCAGCATAACCCTTGTCTATCATGGCTTGAATATTACTACGATACTGAGTTGCATACTCTGGGTCCTTAGCCATCTTTTGTTCTAGAGAAGTAAATCTGCGACGTGCTAGAGCGTAACTATTAGGAAGCTGTCCCAA

ATTAACATCTGACCGCCATAGTAGCCCTGTCTCAAATCGACCAGAAGGGAGTCTGCGTGTGTTGACACGAAGGGTTTCTATGGCGAAAATATCATCCTTGTCAAGTCGCTCTCTCCTATCAATACCAATTGACTCTAGTTTAAAATTTTCTCGAATAAGATTCTCTAGAGAATTATCTCGTACGTGATTGACAGTAGAGTCTGACCGGAAAACTCTACTACAGACGTTCTACTTGTCGGGAAACCGTAAACTACCCATCCTAAAGGAG

TACGACATGCCACAGGCTGAGCGCGTGAGATGCGCCTAACTTCCGACGCTACCAGCAGGTGCCAATTGTCCATGCCGACTAGAATTGATGGTTCCACGTCCGAGTAACTGCACAAATTAAGACCGTTAAAATGCTCCAAATTAGTATCGCATCTCGATAAAGACTGACGAGAGAGCCCAAGATCGCGAACGGAACGCACATGTTTCAAAACATGTGGTTGACAAGTCTTATCATGACCTTTAATTGTAAAGCTAACATACTCCACGTC

TACTCGCTTTTCTAGATTACGCACACATGAGATGATAACACTCTTTTTTAGCCCCGTAACACCGATAGCAGACGAAACCTTAGCGTCTATGAACGTAGCAGTTGAACCCTCATCCAGAAGGGCAAAGGAATCTATGCTGCCACTCGGACCACTGACGGTTACCGGCAAAATCTTAAGAAGTGGTCTGGAATCTTGCTGCTTACATTGATCGCCATCTGTTTCCAAAACTGTCTGCCCCAACGAGTAAACCTGGGTTGAGGAGTCGACT

TTAGACCTATCGTGCAACGCATCGAGTTCGGTAGTCGGTTCTGCAGAAAATTGAGAACCTACAACAGGATCGACATTGGTAGCTGCTTCATGCAAGAGAGAATGGTGGTGACGTTTACGTCGGCAACCGACGACCTTACAACATTTCTTGCAGTTTCTATAGCTGTGACCACCTTTCCTCAAGCATTTGAAGCAAATAAAATTGTCTCTTACCCAACTCAATCGTTTTTCTGCAACCAAATCACGAAACATCTGACACTTCTGGATTT

CATGCGTAGCCTTACAATAAGCACAACCAGGTTCGCTAGAGGTGTTATCTGTGTGACTTGGAAACGATGACTTAGGAACTTTACCTGGTTGTTCGAAGCCTTCTTTGTGTCTCTTGACAGAGCGAAGATGTCCATAATTAGAGACAGGCCTTCCAGTCAAAGTCGTTTGACAGACTGTAGAGGGGTGCTGGTCAAGAGTTGCTGAAGTGAGGGAGGAGTCGACGAAGTCAGCCTGGACAAAATGAGCATGAGACCTATCTGACTTAGG

TTTAAAATCTGTCAGGATACCATATCTTAAGTGCAATAAAGCCTCATGCGATAAGAAATCAGAAAGCTTTTCCAAGCGAAACTTATTTTCATGCTCTTTCTGAAAGGCAAACGCCAACCACTTCTCCCTCAGGAGAGGAGTAAGCTTTCCAAGATAACGTTGCACAGTTCAGGGTTAGCTAAATGGACTTGCTGCTGCAGAAGCTGAACGGTACTTATGCAGTTGCGTACCTTGCATGCAAAAACATTTAGCTCCTGTGCATCGTACC

CGCTTAACAGCGTAGCCGCTTCTGCGCAAGAACGGAAGAACATTTGGTGGCGACTGAATTACCGCTTGAGGAACTCACCTGCGCCCTTTGCTTCGAACGTGACGTGGATGGGGACGGCGGCGGGCCCAACTCTGCGACCGCCGCGGCGGCATTATCCGTAGCGGAGTTAGCCTGCTGCTTTGCGATAGCACGGGTGATCATGCCGCGAACTTTTAAATAACTTCTGACACCAGAAACTGTTAGGTATCGGAGATAAATAAGAAGCCGA

GCTTCCGTCTAACAGTTTTTAATAATTAGTAATAAAAAATCAATGTCAAAAATGGCAAGAAAAGAATATAAGTCAAAAATTAAAGAAAACATAAATGTATCAAAAAGTATGCTACCTACAATGTGTCTATGGCCAGATCTGTAGCCAACAATAGCTATTAATTTTTTTGTTGAATAAAATACTACACATCTACTCTTACCGCGAATCGGACCCTCAGATATGGTCACGAAGGTCGTAAATATTTTTAAGACGTTGTTTCAAACCATCG

CGTTTGAATATAACGTATCATCGTTTGACACCCTTCGGAGAAAAATATTTGCCCAGCAGTGGGAGTCTTTCGACTGAGATGTATACCATGGCTGAAAAAAAGATACTGAATAAGAACACTCGAAAAAAAAAATCGGCATTCGGCGATCTTAACGCTATGCAGCAGTCTCTTTCAGAAAAACTGCATCGTAATAATACTCGTATAATAGAAAAAATCAATCGTTACACAGTTATTTTCAAGAACATTTGTATCATATATTTGTAAATAT

TCTTTTTTATATTGCAAGATCATTTCTTAGTTATTGTTAAATGGTTGTTCCTTTGCGACCCATTCAATGTCTGCGGTCACCAGTCGACCGTTGTTGACAGCGTGTCAGACTGAGAAGGGTCTATCGTTGGAATAACTGACATATTAACACAGTACTTGTACACATGGAAACGATTTTATATAATACGACTAGAATTCTATTGTGATAGTTTTTTTGTTGATGCTAATGAATACCTGGCAGTATCTATAGACGTAGTTACAATTAACCG

TATTTTAACAGAATTAAATTCAAATAAATAAGTGTGCCTATTAGTATTTTTATTAGTTACTAGTGGCTTACCGCGGTTTCACCCGCGCGGTACCCGTTCCAATGGGAATATCGGGATAAAAAGTAGCCTGTATTCTGTATACCAAATTTCAATTAAATTGGTTCATAGTTTTGACGTGAAAGCTAGACTGACAGATAGAGAAATATACAGACAGAGTTACTTTCGCATTTATTATTAAGTATGGATTGTTATTATTTTATTTTAATTT

ATTTACTTGGAAGAATCGTTCACTCAGGTTGAACGTAGAACCCTAATGGACCATCGATTTGTAAAATAATAATAATAATAATATCCTCAGACATTCACACCGATCAACTAGCCCCATTTTAAGCAACTAATGCTGCTTGTGTTACGGACACTAGATGACGGATAAATATATTTAATAGATAATAATAATAATAATAATAATAATAATTTATTTCGATAACTTAGTCTACAATTACAAATTAAATGTGGGATCTCCCTTTTAGGCAAGA

CATCGCCTATAGATAAATACATATAAATAACCAGAATATATAGAAATATATATAAACATCCAAGACTGGAGAACAAATGCCTGAAAATTCATCACCCAAACATTTGCCCCCACTGGGGATCGAACCCAGAACCTCTCGAGATGGCGACGCCTTGAGACCGGCATACAGACCACTCGACCACGGAGGTCGTCAAAAGAACGAGGAAATGCGTTGTATGCGGGCTGCACAGGACCCACCTGCTTAGAGAATTGGAGATCCTTATGGGAGG

CTTGTATTGTATCCAACAGTAGGTGTCTGTCACCTGATATGACATATTGGCGAGTTTAGGTCATTACCTTGGTTTTACATACAAATAACGTATACTCTAAGGTTCCTGAATCTATATAATTTAATTATGAAAATTATCGCATTCGTCTCTGGAATAACCTTCCATTACCAATCAAACAATTCCAATCCTTAGCATCCTTTAATGATCTGGTCCATACCCATTTCCTTCATCTTTGACTCCTTCCTCCAACACACATTGCAGTTTTCGC

CTTTTCTTTGTTGCTTTTTGTGTAAGAATATAGATAATAAGAATATATTGCTAAATAAAATATTTATACAGGATATTGTAGCATTTATAATATAGTATGTATTTTATGTATGTATATAATATATTATGTAATTTATATGTAGTAGTTTATATTTGCACCTTATATATGTTACCTACCTTTTCTTTCATTCTATCTCTGTCCAAAGGTTGTCTGGAAGATATTGCTATTAGCTATAAGGCCGCCTGTGTTGTATTGTATTTTTTTACTA

TATTTCTGTTTTAATTAGTTGCAATAAAGAATTTTATCTATCTATCTATTGCCGATAACTTTCATGAAGAATTTTCCTATTCATGAATACCATGAACAACAAACCTAAACTCACCCTTCTCACTCAAGTTAACTTATGCCAACTATAAATAATAAAAATAAACGAAACCCACATTATGATTGTGTAGTAAAATCTGACAGTAAATTAATATTTCTATTTGGTAAGGCCGGTACATTTAAAGCGTTTTGTTTTAAAAATACAGCTGGCT

GTGTGCCAAGCAAGCGAATACTATATTGGGCCAGCCTGGCTAATTGTTACTTCTGAATGCAACGAGGGAAATTTCTACAGCCTCCGACAACATTGTGGATAATTTTTCGACTGTATCCAACGTTGTAGTTCCTTTACAAGAATACAAAAAGCTCTTTAAACCTATTATGAGTCCTGTGCGCATTTCGGAATTTCGAGTTGCGTTCGAGTTTTTCACTTGTTTTACCAGGAAGTTATTGTAGGGATGACTGTAATTATGATACAAACAT

TTTTGATGATGTTGTAAAAAAAAAGCTATTGCATAGCATTTTGTATCAACTTATGCAATTATAATTTTATTTATTTATTTTATTCATGCAGTATTTCTTTTTCTTTTTTATTATTTTACAATACATGTGGGTTTTATTATCTTAACGCCGTGTCGAAGACTGCCGAACTGAAACGACGTTAGACGATGCACGGCCTTCAAGCCACACCTCCTTGCGCATCGGAGTGGGGAGCGTGAGGTTTTTTCGTTACGGAATTTCTGGATTCGGT

CCCCGCGCTCAAGGCCCGAGATAGAAGCTATGCAATAGCTTAAAAGCTTGTAGAGAAAGAGATAAAACGCCTAAAAATGGAGACCTAACATATCAGCAAAATATGGGTGATAACACCTCCCTACCCCTTTGCAGAAAGAGGCGCGATGATATGTTATTCCATATTTTTTTTACATATTTACATGCAGTGTTATTGTAAATGGACAACCTCCGTAGCCGAAATTTGTACGCTGGTTCGAAGGAATCTCGTCATTTTTAGAGGTAGGCTC

ATTTGATCTTCGGTGATTTATTCTCTAAAACTTATATGTATTGAATATTATGGGCCCTGGCAGGGAAAGTACCTTACATATTTAAAATAGATAAATTTACATAAAGGACTAACTTTTGTTTTTAATAATTAACATAACTGATCTCTCAGTTTTTTTTTAAAAAAGCTTACCCAAACCTTCCCTGGAATATAAAATAATTGTTAGACATGTTTAAAACGTAAAGCTTAATAAAGAAATTAAATTTTCAACAATGACTTAATCGGTTAGT

GGAATTAAATCGCGAACTTTTTTTATTCATTTTTTCCTTATTCGATTCTCGGTTTGAGTAAGCTAGCTAGCTATTTTGAGAATATTTAGAACTTAGTTATGTTTCTTATTCAAAGTAGAAGTATGCTCTTGTGTAAATTATATTTTTTTTTAATATTAGCTAATAGCTCCAGGGCCCACAAAATTTTCCACTCTGTATGTAGGTATATACTTATCTATTACGCTCATTAAAACTTATCCGTCATTTAGTATCCATAACACAAGCAGTA

TAAGCTGCTTATTTTAGGCTACTTGATAGGTGAAAAAAATATGTATTACATTCATACATACATTCACGCCTCTTTCCCAGGGGGGTTGGCAGAGATTACAGATCTGCACTTGCTACGATCCTGCATCCCGCACATGACACACACTGTCTCTACCCTCATTACTAACTTTATACATGCCCTTCGGTTAATTTTAATAATTATTTATTAGAATATTAAGCTTTATTATGATTAATAGCGATGGCAATTCGTTAGCTTATAGGCGCCATTT

TCTTTATCCTCTACTTAAGTAGTAGCCAGCAATTTTAGGGCAGTGACCTAAGGTCAACGACTTGTAAAGGATAGTTCTTATGTTTTAAGAATACCGCATAGTAGATACGAATAACGTTGCAGTCTAACCGTGCAGAAGATTTTTGTCTTTCCGGATGATTAGATGTTATGAATATGAGGGAAAATATGCTTTAAGTATGAAGACTATAAGGTCTAGTATTTAATTTGATGTTATAGTTATCTTCGCACTGCTCTATAGATTCTTTTAT

ATAAAAATGACTTTTATTTTTTTATTAACAGCAACATTCTAATCTAAAATAATATAGACCAATTTTATATATTTACTTTTATATTTACCTGTTAGATTGAGACTGAGAAATATCAGTCGAATGGGAGTGTTTAAAACGCCTGTCTACCCCTTTGGGATAGAGGCGTACGTTTATGGTTTATTTTGATGTTTGTTGTATGACATATTACACTAAATCTCTACAAAGAAGAACAAGACGAGCCGAGTTGCCATGCATCGGGCACAAAGTG

AAGCAAGCAATATTAACAATGAACTGTACATAACTTTTAATTCAAACTCGGTAGAACTCGCGAATCCCGCGGCCTCTCCAAGGAGTAGTGCCCTCTGTCCCAATTCAGCTAAGGAATAGTCAACGTAATTACTAAAGCAGACTTATTGTCTCCACCACATGTAAACCGTTTGAATCATGCATTAATAAAACTTCATGTCCCCAACAGTGGGCACTGGCTTTTACCTGACTTCATATCTTACACATTTATATCCTTAGAGGAGTATACC

GAGGGGTCATGATTTTTGTAATCTAACAATGTATGTTTGTATTTTAAATTGATAATCCAGACTTATTATAAATGCGAAAGTAACTCTGTCTGTCTGTCTCGCTTTCACGTCAAAACTACTGAACCAATTTAACCGAAATTTAGTACACAGATAGTCTAGAACCTGAGAAAGGACATAGGCTACCATTTATCCCGATATTACCATGGGAACGGGTGCCATAGGGGTAAATGTAAAAACATTTAAATAACGCCTCCCAGAAAAATGAAGG

ACAAATTTACGCCACTATGAACTTTGCTTCTAATAATAGAGTAGGAGTAAATTTTCAATTCCACCAATTGAGGCTACGGTTCTTATTTTAAAAAATCTTTCAGTGTTCAATAGCCTATTTTTTGAGGAAGGCTATAGGCTACATACCATTAATCACACTATCTCAAAGTCACTATTCTACGCGGATGAAGTTGCGGGCAAAAGCTAGTTGATAAATTCATTGATGTATTGATAATTCATAGACCATCAAGTCATTTAAAAACGTAATA

AAAAAAAATGTGTTAAAAGCCTCAAAAAAAATTATATAATTTTTTATGCTGGTGGCTAACAGAAATGAGTAATTTGGGGCCCAATAAAAAAAGGGTCAACTAGCCTAATATTTTAATTTCTTTTTTGTACTCATTATATTAGGTCCTTTCCTATGAAATTGGTGTTTTGTATGGAGATTTTGGGGATGTCTGCATTTTAAATACTAACATTTTATCAGTTTTCTTCACAACGACTAATCCAGAGATAATAATATTTGGCATGTATTAT

AAGACAAGCTTTAGCAATATGTGTGTGAACACGCTTGATAAAAATTATTCATCGTTTTTATGTTAATGCAAATCAAATGATGTATTCGGGAGGCAGTTAACGTTTAAACATCAGCCTATGTTTGTCCACTGCTGGACATAGGCTTACCCTAATGCATCGAGAGGTTATTCGAAAGATTAATTGCTGTTTAGCTGAAAGGTAAATTGTGAAATCAATCTAGTTTCAAGAAATTAACTGTTTATCCTATGTGGAGTTATCAGTAAAATGT

TTTTCCACTAAATTGTTAGTTAAAATTTTACACAGCAATCCAACATAACTCTTCTAGTCATAGCAGCAGAGTGGTTTTTTAAAAACCGACCGTTTGTTGCCGACACCACACCTGTTTCGCAATTTTTACAACATTCAAAACACCACAAACTCATTTAAACTACACTACATCAAATTGTCACGTTAAATTTATTACAATAGAGTTGCTTTTTGTTACAAAAACCTTTAAAAATGTATAGTTTTACGATATTGAACTTTAAAGCGGTAAT

AATAAAGCCTTAATATTGTGTTTTAAGACTATTATCTAACGACGCCGTTCGCGTAATTTTAGTTACATTGATCCAATTAATATGAAAACTTATAGCTGCTATCGTGTTTCGACACTAGATTTTCTTTGACACCAAAAAAGTACTAAGCTTATTACATAAAAGGCCAAATAGTGTTATAATTGTTTGTTTTGACAATCACTGATGAAACTTTGATATATTTTTCCCGATGTAGGTATTCGTCTTAGTACACTACTTAAATAAATAGAAC

ATTTTACTAAAATGTTCTTCATTGTAGTAATTATCGCTATTTCTTTTGAATCAAAAATTTAATAATATTAACGCGACGTTTCCCGATCACGCTATATTCCCGAATGGGTAGTTTTAATCTGAGCAACTTTACAATAATAACTTTTTAAATCATAGTTGATTAATAAATAAATTATTCAAAAAAAAATACAAATTTAAGACTAAAAATACTTAAGACAAAATTATACTTACAGACTTTTTATTATCATTT

CIRCULAR CONSENSUS SEQUENCING (CCS) MODE:20 KB HUMAN HIFI LIBRARY – READ LENGTH AND RAW BASE YIELD

PERFORMANCE

Data shown above from a 20 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (2.0 Chemistry, Sequel II

System Software v8.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample

quality/type and insert size.

METRICS

Insert Size 20 kb

Number of Raw Bases 377 Gb

Total Reads 4,266,403

Half of Bases in Reads (bp) >193,403

Longest Read Lengths (bp) >300,000

Customer Average:

325 Gb Raw Bases

METRICS

Insert Size 20 kb

Number of HiFi (>Q20) Bases 26 Gb

Number of HiFi (>Q20) Reads 1,423,277

Mean HiFi Read Accuracy 99.92%

CIRCULAR CONSENSUS SEQUENCING (CCS) MODE (CONT.):20 KB HUMAN HIFI LIBRARY – SINGLE-MOLECULE ACCURACY AND

HIFI BASE YIELD PERFORMANCE EXAMPLE (POST-CCS PROCESSING)

Data shown above from a 20 kb size-selected human library using the SMRTbell Template Prep Kit on a Sequel II System (2.0 Chemistry, Sequel II

System Software v8.0, 30-hour movie). Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample

quality/type and insert size.

≥Q20 (99%)

HiFi accuracy

WHOLE GENOME SEQUENCING APPLICATIONS POWERED BY HIFI

READS

De novo assembly of genomes with high

contiguity, correctness, and

completeness

1 Human Genome

per 2 to 3 SMRT Cells 8M(~70 – 100 samples / yr / Sequel II)

High precision and recall for single-

nucleotide variants, indels, and structural

and copy number variants with HiFi reads

1 Human Genome

per 2 SMRT Cells 8M(~100 samples / yr / Sequel II)

- For researchers who want to detect variants comprehensively in a

whole human genome, the Sequel II System provides high precision

and recall for single-nucleotide variants, indels, structural variants,

and copy-number variants, including in repetitive regions of the

genome.

- HiFi reads combine the benefits of high accuracy and long read

length.

- Recommend using 2 SMRT Cells 8M to achieve ~15-fold HiFi read

coverage of a whole human genome for comprehensive variant

detection

- Variant calling with HiFi reads can be performed with standard

software tools like Google DeepVariant and GATK.

- Run ~100 human samples (3 Gb) per year, per Sequel II System*.

VARIANT DETECTION APPLICATIONS

POWERED BY HIFI READS

Gain complete views of human genetic

diversity with PacBio long-read sequencing

* Read lengths, reads/data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size. Prices, listed in USD, are

approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument amortization or

other reagents.

HIFI READS ENABLE HIGH PRECISION & RECALL FOR DETECTING

ALL VARIANT TYPES

Variant calls from ~15-fold HiFi read coverage of a human genome (HG002) were measured against the Genome in a Bottle small variant benchmark

(v3.3.2) for SNVs and indels using Deep Variant and SMRT Link 8.0 for SVs. Libraries were generated using a 15 kb insert and sequenced using

Chemistry 2.0.

Perc

en

tag

e (

%)

99.1

99.6

95.0

96.6

97.7

95.2

90.0

91.0

92.0

93.0

94.0

95.0

96.0

97.0

98.0

99.0

100.0

Recall Precision Recall Precision

SNVs Small Indels SVs

Recall Precision

15-FOLD HIFI READ COVERAGE RECOMMENDATION FOR

COMPREHENSIVE VARIANT DETECTION APPLICATIONS

15-fold HiFi (≥Q20) Coverage[2 SMRT Cells 8M for a 3 Gb genome] provides a good trade-off between cost and results

Wenger, A. et al., Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. 2019. Nature Biotechnology.

- HiFi reads provide assemblies with high contiguity, high base

pair accuracy, and high gene completeness.

- With megabase size contig N50s, accuracies >99.99%, and phased

haplotypes, you can do more biology – capturing undetected SNVs,

fully intact genes, and regulatory elements embedded in complex

regions.

- Compared to using standard PacBio Long Reads (via CLR

sequencing mode), de novo assembly using HiFi reads

generated with the Sequel II System affords smaller file sizes

and faster analysis times.

- Assemble up to a 2 Gb genome in a single SMRT Cell 8M for

~$1,300*.

- Run ~200 samples (2 Gb) per year, per Sequel II System*.

DE NOVO ASSEMBLY APPLICATIONS

POWERED BY HIFI READS

* Read lengths, reads/data per SMRT Cell 8M, and other sequencing performance results vary based on sample quality/type and insert size. Prices, listed in USD, are

approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument amortization or

other reagents.

PacBio is the only single-technology solution

for easy and affordable generation of high-

quality de novo assemblies.

GENERATE CONTIGUOUS DE NOVO ASSEMBLIES USING HIFI

READS

DATASET RICE DROSOPHILA HUMAN

HiFi Library Insert Size 17 kb 19 kb 15 kb

HiFi Read Coverage 20-fold 20-fold 22-fold

Contig N50 (Mb) 10.7 6.5 30.5

Obtain high contiguity assemblies with HiFi data; high

accuracy allows assembly through very similar repeats

GENERATE COMPLETE AND ACCURATE DE NOVO ASSEMBLIES

USING HIFI READS

Accuracies >Q40 (99.99%)

>98% of genes in frame

DATASET RICE DROSOPHILA HUMAN

Assembly size (Gb) 0.400 0.150 2.92

Base pair accuracy

(Phred/Percentage)Q50 / 99.999% Q50 / 99.999% Q49 / 99.9987%

BUSCO completeN=1,440

98.7%N=2,799

98.9%N=4,104

94.9%

Species-specific

genes in frame

N=19,313

98.7%N=19,947

98.9%N=35,666

99.5%

GENERATE DE NOVO ASSEMBLIES QUICKLY WITH HIFI READS

Compute times for de novo assembly of a human genome

DATA TYPE HIFI READS LONG READS

File Type CCS.FASTQ.GZ SUBREADS.BAM

File Size (GB) 48 323

Read Correction Method CCS Analysis Pre-assembly

Time to Results

(Hours)

Read

Correction17.5 43.5

Contig

Assembly13.7 18.9

Analyses run with PacBio recommended compute

infrastructure~31

hrs

~62

hrs

Assembly with HiFi Reads takes half the time of

assembly with Long Reads.

COMPARISON OF DE NOVO ASSEMBLY RESULTS FOR CALIFORNIA

REDWOOD TREE (ESTIMATED 27 GB HEXAPLOID GENOME)

[1] Hybrid assembly of redwood using ONT + short reads (2019).

[2] Douglas Fir assembly by Neale et al. using short reads (2017)

[3] Transcript set of Abies alba from Neale et al. (2019). Varying number of transcripts aligned to each genome (4,958

mapped to PacBio HiFi redwood, 4,760 mapped to ONT redwood, 16,187 mapped to Douglas fir)

PacBio’s HiFi de novo assembly of the large California redwood (Sequoia sempervirens)

achieves markedly improved performance in the 3 C’s of genome assembly quality (Contiguity,

Completeness, and Correctness) compared to previous conifer assemblies using other

technologies.

A 24Kb PacBio HiFi Library was prepared and sequenced and the raw HiFi data was accessioned and deposited in NCBI under BioProject PRJNA606797.

HIFI SMRTBELL EXPRESS 2.0 LIBRARY PREPARATION &

SEQUENCING WORKFLOW SUMMARY OVERVIEW

1. gDNA QC & Shearing

- CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC

- Shearing with Megaruptor or g-Tubes

2. SMRTbell Express Template Prep Kit 2.0 Library Construction

- Perform single-tube, addition-only reactions

3. Size-select & Purify SMRTbell Library

- Perform size selection using SageELF for HiFi Variant Detection or BluePippin for HiFi de novo

Assembly applications

4. Sequencing Preparation (CCS Sequencing Mode)

- Perform Sample Annealing / Binding / Cleanup (A/B/C)

- Follow QRC for loading recommendations

5. Analyze

- For variant detection analysis, can use GATK or Google Deep Variant

- For large genome de novo assembly, can use Falcon-Unzip or other third-party software

Workflow summary for constructing SMRTbell libraries ~11 – 20 kb (or larger) that

are suitable for generating high-accuracy long reads on the Sequel II System using

SMRTbell Express TPK 2.0 for de novo assembly and variant detection applications

~4.5 hours

HiFi Library Sample Preparation Workflow

Details

PROCEDURE & CHECKLIST – PREPARING HIFI SMRTBELL

LIBRARIES USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0

- Procedure & Checklist describes a method for

constructing SMRTbell libraries ~11 – 20 kb (or larger)

that are suitable for generating high-accuracy long

reads on the Sequel II System using SMRTbell Express

TPK 2.0 for de novo assembly and variant detection

applications

- Protocol document contains:

1. Recommendations for gDNA QC and quantification

2. Recommendations for shearing gDNA to the desired

target mode size using either the Megaruptor System

(Diagenode) or g-Tubes (Sage Science)

3. Enzymatic steps for preparation of a HiFi SMRTbell

library using SMRTbell Express TPK 2.0

4. Instructions for size-selection of the HiFi SMRTbell library

using either the SageELF System (Sage Science) or

BluePippin System (Sage Science), and also includes

protocol reference for performing AMPure BP Size

Selection method for de novo assembly applications

using HiFi reads if required

5. Sample setup guidance for preparing HiFi libraries for

sequencing on the Sequel II System

https://www.pacb.com/support/documentation/

HIFI SMRTBELL EXPRESS 2.0 LIBRARY PREPARATION &

SEQUENCING DETAILED WORKFLOW OVERVIEW

1. gDNA QC & Shearing

- CHEF Mapper, Femto Pulse or Pippin Pulse sizing QC

- Qubit DNA concentration measurement QC

- Shearing with Megaruptor or g-Tubes

2. SMRTbell Express TPK 2.0 Library Construction

- Perform single-tube, addition-only reactions

- ~4.5 h library construction workflow followed by size-

selection

- Typical library yield ~50%

3. Size-select & Purify SMRTbell Library

- Perform size selection using SageELF for HiFi Variant

Detection or Blue Pippin for HiFi de novo Assembly

applications

- (Optional) alternative AMPure PB Size Selection method

is available for de novo Assembly applications (Note:

HiFi read lengths will be shorter)

4. Sequencing Preparation (CCS Sequencing Mode)

- Anneal Sequencing Primer, bind Polymerase, perform

AMPure PB complex cleanup

- Follow QRC for diffusion loading recommendations

- 2 hrs (≤20 kb) / 4 hrs (>20 kb) Pre-Extension Time; 30-

hour movie collection time (Sequel II System)

5. Analyze

- For variant detection analysis, can use GATK or Google

Deep Variant

- For large genome de novo assembly, can use Falcon-

Unzip or other third-party software

1

2

3

5

4

Day 1

Day 2

PROCEDURE & CHECKLIST – PREPARING HIFI SMRTBELL

LIBRARIES USING SMRTBELL EXPRESS TEMPLATE PREP KIT 2.0

List of Required Materials and Equipment for Library Construction and Size Selection

LIBRARY CONSTRUCTION WORKFLOW RECOMMENDATIONS FOR

SPECIFIC HIFI SEQUENCING APPLICATIONS

- High-quality genomic DNA (gDNA) can be sheared using a Diagenode Megaruptor tool (up to ~100 kb

fragment size) or Covaris g-TUBEs (up to ~20 kb fragment size)

- Depending on project requirements, SMRTbell libraries are size-selected using a Sage Science

SageELF system (recommended for variant detection) or BluePippin system (recommended for de novo

assembly). Note: AMPure PB Bead size selection may optionally be used for de novo assembly

applications but may result in shorter mean HiFi read lengths (compared to using BP size selection)

LIBRARY CONSTRUCTION RECOMMENDATIONS FOR APPLICATIONS REQUIRING HIFI LONG READS

HIFI

APPLICATION

FOCUS

SIZE-SELECTION

METHOD

NUMBER OF COLLECTED

FRACTIONSNOTES

HiFi Variant

Detection

SageELF

(Recommended)

5 Fractions

(~11 kb, ~13 kb, ~15 kb, ~17 kb, >19 kb)

Resulting HiFi reads may also be used for de

novo assembly.

BluePippin2 Fractions

(~11-13 kb, ~13-20 kb)

Resulting HiFi reads may also be used for de

novo assembly.

HiFi de novo

Assembly

BluePippin (High-

Pass)

(Recommended)

1 Fraction

(>15 kb)

Removes <15 kb SMRTbells from final library

(with no upper-end MW cutoff).

Resulting HiFi reads are not recommended for

variant detection.

AMPure PB Beads1 Fraction

(>~5-20 kb, depending on shear distribution)

Removes <5 kb and reduces <10 kb SMRTbells

from final library

Resulting HiFi reads may show shorter HiFi

read lengths (compared to BP size selection)

and are not recommended for variant detection.

GENOMIC DNA SAMPLE INPUT & QUALITY REQUIREMENTS FOR

HIFI SEQUENCING

- This procedure describes construction of HiFi libraries from gDNA with a sheared mode size of >15 kb

- Table below summarizes DNA input, quality and DNA shear mode requirements for specific size-selection

options.

- The final SMRTbell library synthesis yield (%) of the collected and purified HiFi fractions depends on the

quality of the starting genomic DNA and distribution of the DNA shear.

- To increase the recovery yield of larger fraction sizes (>20 kb), the target shear size distribution

must be adjusted so that the mode is 20 kb or larger.

- Always perform test shears prior to starting SMRTbell library construction.

DNA INPUT REQUIREMENTS AND RECOMMENDED SHEARING METHODS FOR CONSTRUCTING HIFI SMRTBELL

LIBRARIES

Size-Selection

Method

Required Input gDNA

Amount

Required Input gDNA

Quality (Mode Size)

Target Sheared

Fragment Size

Distribution Mode*

Shearing Method**

SageElf 15 µg >40 kb >15 – 20 kb (or larger) g-TUBE or Megaruptor

BluePippin 15 µg >40 kb >15 – 20 kb (or larger) g-TUBE or Megaruptor

AMPure PB 15 µg >40 kb >20 kb g-TUBE or Megaruptor

* To increase the recovery yield of larger fraction sizes (>20 kb), the target shear size distribution must be adjusted so that the

mode is 20 kb or larger.

** gDNA can be sheared up to ~20 kb fragment size using Covaris g-TUBEs

1. CHEF Mapper XA System (Bio-Rad)

2. Femto Pulse System (Agilent)

3. Pippin Pulse System (Sage Science)

http://www.sagescience.com/products/pippin-pulse/

http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems

https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-

systems

➢ Up to 10 Mb

➢ >16 Hour Run Time

➢ Up to 80 kb

➢ 16 Hour Run Time

Recommended methods for determining gDNA size distribution:

Lane 1: 8-48 kb Ladder (Bio-Rad)

Lane 2: 5 kb Ladder (Bio-Rad)

Lane 3: HMW gDNA

Lane 4: Degraded gDNA

Lane 1: High MW gDNA

Lane 2: Degraded gDNA

Lane 3: 165 kb Ladder

Evaluation of gDNA quality using A) Bio-Rad CHEF Mapper and

B) Advanced Analytical Femto Pulse. Lanes A3 and B1 show an

example of a high quality, high molecular weight gDNA sample,

where most of the DNA migrates as a prominent band at the top of

the gel image. Lanes A4 and B2 show an example of a partially

degraded gDNA sample where most of the DNA migrates below ~40

kb and is thus not suitable for constructing a >15 kb HiFi SMRTbell

library.

A B

RECOMMENDATIONS FOR EVALUATING GENOMIC DNA QUALITY

➢ Up to 165 kb

➢ 1.5 Hour Run Time

Femto Pulse System only requires

~0.5 ng of gDNA sample and

assay run time is <1.5 hours

EXAMPLE SIZING QC ANALYSIS OF STARTING INPUT GENOMIC DNA

ISOLATED FROM A TREE PLANT SAMPLE

- Use these example size distribution profiles as guidance for determining the appropriate starting amount of

gDNA to use for this procedure. E.g., if your starting gDNA fragment size distribution is significantly lower in

quality than shown here, consider increasing the amount of starting input gDNA amount to be higher than

15 µg to help achieve optimal library construction recovery yields.

Size distribution mode of starting gDNA should be >40 kb to help achieve optimal

library construction recovery yields

Femto Pulse sizing QC of replicate Redwood tree plant genomic DNA samples. gDNA was isolated using the Circulomics Nanobind

Plant Nuclei Big DNA Kit (PN NB-900-801-01).

Unsheared

gDNA (160 kb Mode)

>40 kb

PERFORMING ACCURATE SIZING QC THROUGHOUT HIFI SMRTBELL

LIBRARY CONSTRUCTION IS IMPORTANT

SMRTbell library (17.3 kb Mode)

Sheared gDNA (19.8 kb Mode)

- Use these example size distribution profiles as guidance for determining the appropriate starting amount of

gDNA to use for this procedure

- To increase the recovery yield of larger HiFi SMRTbell insert sizes (>20 kb) during the size selection

step, the target shear size distribution must be adjusted so that the mode is 20 kb or higher.

Size distribution mode of sheared gDNA should be >15 kb to help achieve optimal

library construction recovery yields

Femto Pulse sizing QC of a sheared human gDNA and SMRTbell library sample. gDNA was sheared with the Megaruptor 1 System.

>15 kb

GENOMIC DNA SHEARING RECOMMENDATIONS FOR HIFI

SMRTBELL LIBRARY CONSTRUCTION

This Procedure & Checklist describes the construction of HiFi libraries from

gDNA with a target sheared mode size of >15 – 20 kb or larger

- Genomic DNA can be sheared using a Megaruptor System (Diagenode) or g-TUBEs*

(Covaris)

- Note: gDNA can be sheared up to ~20 kb fragment size using Covaris g-TUBEs

- If the goal of your project is to sequence larger HiFi library fraction sizes (>20 kb), the

shear size distribution must have a mode of 20 kb or larger.

- Megaruptor System is recommended if shearing gDNA to a target sheared mode size >20 kb

- The response of individual gDNA samples to recommended shearing parameters may differ,

so small-scale test shears are highly recommended.

- Under- or over-shearing gDNA will result in low yields of the final, size-fractionated library.

- For high quality gDNA, typical yields of sheared and concentration DNA are approx. ≥70%.

- Because you will need 10 μg of sheared gDNA for the subsequent enzymatic steps, PacBio

recommends starting the shearing procedure with at least 15 μg of input gDNA.

* See Appendix I of this technical training presentation for an overview

of an optional alternative shearing method using Covaris g-TUBEs to

shear gDNA samples to a target mode size of ≥15-20 kb.

MEGARUPTOR TOOL RECOMMENDATIONS FOR SHEARING

DNA FOR HIFI SMRTBELL LIBRARY CONSTRUCTION

Megaruptor generates a tight DNA shearing distribution profile that results in good

recoveries during SMRTbell library size selection

- Dilute 15 µg of high molecular weight gDNA in 1x Elution Buffer to a

concentration of 50 ng/µL in a volume of 300 µL

- It is important not to exceed this DNA concentration during shearing

or you may clog the hydropores

- Before shearing, remove a 1 µL aliquot (un-sheared sample) for QC

- Shear gDNA to the desired target mode size with long hydropores

using the recommended settings in the Megaruptor software as

shown in the Table below

DESIRED TARGET SHEAR

SIZE MODEMEGARUPTOR SYSTEM

MEGARUPTOR SUGGESTED

SOFTWARE SETTING*

11 – 13 kb Megaruptor 1 or 2 15 kb

15 – 18 kb Megaruptor 1 or 2 20 kb

21 – 23 kb Megaruptor 3 Speed 30

25 – 27 kb Megaruptor 3 Speed 28

* Perform small-scale test shears to verify appropriate Megaruptor software settings to achieve the desired target shear size mode for a specific sample.

Megaruptor 2 Megaruptor 3

RECOMMENDED MEGARUPTOR SOFTWARE SETTINGS FOR SHEARING GENOMIC DNA FOR HIFI LIBRARY CONSTRUCTION.

MEGARUPTOR TOOL RECOMMENDATIONS FOR SHEARING

DNA FOR HIFI SMRTBELL LIBRARY CONSTRUCTION (CONT.)

- Run time is approximately 15-20 minutes per tube using a Megaruptor 1 System.

- Proceed to the next step to concentrate your sheared gDNA using AMPure PB beads

- Note: The Megaruptor 1 tool dilutes DNA during shearing, so empirically measure the

volume of your sheared gDNA sample before AMPure purification

- Because the response of individual gDNA samples to recommended shearing parameters

may differ, small-scale test shears are highly recommended (e.g., 50 μL at 50 ng/μL)

- Under- or over-shearing gDNA will result in low yields of final, size fractionated SMRTbell

library

PERFORMING SHEARING OPTIMIZATIONS IS IMPORTANT

.

Sample 1

12.3 kb Mode

Sample 2

10.3 kb Mode

Sample 3

13.5 kb Mode

- Above Figure shows examples of 3 different gDNA samples sheared using the same Megaruptor 1 System

shearing protocol.

- Under- or over-shearing impacts yield of fractions recovered from the library size selection step, hence

impacting the number of SMRT Cells that can be achieved per library prep

Performing test shears is recommended since different genomic DNA samples may

shear differently

MEGARUPTOR 1 SYSTEM HUMAN GENOMIC DNA SHEARING

EXAMPLE

- Femto Pulse analysis of a human gDNA sample sheared to a 18 kb mode size using the ‘20 kb’ shear size

setting in the Megaruptor 1 software.

Sheared gDNA

(18.2 kb Mode)

MEGARUPTOR 3 SYSTEM BACTERIAL GENOMIC DNA SHEARING

EXAMPLE

- Femto Pulse analysis of replicate E. coli gDNA samples sheared using the ‘Speed 30’ setting in the

Megaruptor 3 software. Sizing QC results indicate that the gDNA was sheared to a ~22 – 23 kb mode size.

Sheared gDNA

(Mode = 22 – 23 kb)

MEGARUPTOR 3 SYSTEM PLANT GENOMIC DNA SHEARING

EXAMPLE

- Femto Pulse analysis of replicate Redwood tree plant gDNA samples sheared using the ‘Speed 28’ setting

in the Megaruptor 3 software. Sizing QC results indicate that the gDNA was sheared to a ~25 – 27 kb

mode size

Sheared gDNA

(Mode = 25 – 27 kb)

SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR VARIANT DETECTION APPLICATIONS

- 12 size fractions are collected per SageELF gel cassette

(Run Time is ~4.5 hours)

- SMRTbell libraries with insert sizes of ~15 – 25 kb are

suitable for HiFi sequencing

- SageELF Cassette Definition File and Run Protocol Setup

(Pages 13 – 14 in Procedure)

- “0.75% 1-18kb v2”; ‘Size-based’ separation mode; Target

value = 3400 (move the bar slider to select Well #12)

- Recommended SMRTbell library input amount per lane: 5 µg

- For >5 µg, use a second gel cassette

- Typical recovery yields for each collected fraction of interest is ~≥10% of the total amount of library

material loaded per lane – but will be highly dependent on the size distribution of the starting sheared

gDNA

- Recommend performing DNA quantitation and sizing QC analyses for samples collected in

the following wells:

- 15 – 20 kb HiFi Libraries: Wells 3, 4, 5, 6 and 7

- >20 kb HiFi Libraries: Wells 1, 2, 3, 4, and 5

- Purify each size-selected fraction of interest with 0.5X AMPure PB Beads

- Final elution volume is 11 µL

- Perform final sizing QC for each purified fraction (e.g., by Femto Pulse, Fragment Analyzer,

CHEF Mapper or Pippin Pulse)

- Save desired fractions for sequencing (or store fractions at -20°C for future use)

* See Appendix II of this presentation for an overview of an optional alternative

size selection method using BluePippin (Sage Science) for variant detection

applications.

SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR VARIANT DETECTION APPLICATIONS (CONT.)

SageELF SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR VARIANT DETECTION APPLICATIONS (CONT.)

Left figure shows an overlay of Femto Pulse electropherograms for SageELF Fractions 2, 3, 4 and 5 collected for a human library

sample. Fraction 3 contains size-selected SMRTbell templates with a mean insert size of ~15 kb.

F2

F3

F4F5

F3

Fraction 3(15.2 kb Mode)

F4 F2F5

15.2 kb10.7 kb 17.9 kb9.4 kb

Always perform sizing QC on recovered SMRTbell library fractions after size-

selection

- To determine which fractions are suitable for generating HiFi reads, perform a sizing QC on

all recovered SMRTbell library fractions of interest after size selection using Femto Pulse,

Fragment Analyzer, CHEF Mapper or Pippin Pulse

- Femto Pulse sizing QC method is highly recommended since it requires very small amounts of DNA

sample (≤500 pg)

- SMRTbell libraries with insert sizes of ~15 – 25 kb are suitable for HiFi (CCS) sequencing

SIZE SELECTION WITH THE BLUEPIPPIN SYSTEM

- BluePippin size selection protocol options for HiFi library construction:

- Option 1: Size-select a single fraction (>15 kb) – Recommended size-selection method for de

novo assembly applications [see Slides 41 – 44 in this presentation for description of procedure]

- Option 2: Size-select two fractions (9 – 13 kb and >15 kb) – Can be used to size-select HiFi

libraries for variant detection applications if a Sage Science SageELF system is unavailable [see

following Slides 67 – 71 in Appendix II of this presentation for description of procedure]

- For the latest BluePippin User Manual and guidance on size-selection protocols, please

contact Sage Science (www.sagescience.com)

- Note: Visit Sage's website (http://www.sagescience.com) to verify that your BluePippin

software is up-to-date.

As an alternative to the SageELF system, Sage Science’s BluePippin tool may also

be used to size-select libraries for HiFi sequencing

BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS

1. Prepare up to 5 µg of SMRTbell templates in a final volume of 30 μL Elution Buffer for

each Blue Pippin lane.

2. Bring the Loading Solution to room temperature, and then add 10 μL of the Loading

Solution to the 30 μL DNA sample. For loading multiple lanes with the same sample,

scale the volumes proportionally. The Loading Solution is viscous, so pipet slowly to

ensure complete transfer into the DNA sample.

a) Pipette mix using wide-bore pipette tips to mix.

b) Spin briefly to collect the contents at the bottom of the tube.

3. Follow the manufacturer’s recommendations to set up a run protocol.

a) Select the “0.75% DF Marker S1 high-pass 15 kb -20kb” Cassette Definition File for your

sample.

The following BluePippin High-Pass procedure may be used to size-select a single

fraction (>15 kb) for de novo assembly applications using HiFi sequencing

BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS (CONT.)

3. (Cont.)

b) Using the “Range” selection mode, enter a desired “BPstart” value (e.g., between 15 kb and 20 kb).

A “BP End” value should automatically appear.

Note: Sample lanes containing <3 µg of SMRTbell library material will run faster during

electrophoresis. In such cases, PacBio recommends adjusting the BP Start values as follows:

* PacBio does not recommend running BluePippin size selection with <2 µg of SMRTbell library material per lane.

c) Be sure to assign a marker lane. We recommend using Lane 4 for the marker.

4. Load the S1 marker and samples into the BluePippin gel cassette and start the run. Run

time is approximately 4.5 hours.

5. Collect the size-selected library: To maximize recovery of eluted DNA, wait at least 20

minutes after the run terminates before removing the sample from the elution chamber.

a. Collect the eluate containing the size-selected library into a 2.0 mL DNA LoBind tube.

b. Wash the elution well with 40 μL of Sage Science’s 0.1% Tween-20 Wash Solution and then add the

recovered wash liquid to the eluted sample. Washing the elution well may further increase recovery

yields by approximately 10-20%.

CASSETTE DEFINITION FILE IF < 3 µg INPUT PER LANE*, USE BP START

0.75% DF Marker S1 high-pass 15 kb -20kb

12500 for 15 kb cutoff

13500 for 17 kb cutoff

15000 for 20 kb cutoff

BLUEPIPPIN SYSTEM IS RECOMMENDED FOR HIFI LIBRARY SIZE

SELECTION FOR DE NOVO ASSEMBLY APPLICATIONS (CONT.)

Sheared gDNAMode = 27 kb

SMRTbell Library

(Pre-Size Selection)Mode = 24 – 25 kb

Example Femto Pulse Sizing QC for >17 kb BluePippin Size-Selected HiFi Libraries

SMRTbell Library

(Post-BP Size Selection)Mode = 23 – 25 kb

Femto Pulse sizing QC analysis for replicate Redwood tree plant library samples size-selected on the BluePippin system using

the “0.75% DF Marker S1 high-pass 15 kb -20kb” cassette definition file with a >17 kb lower molecular weight cutoff. Most

SMRTbell templates <17 kb are removed after BluePippin size-selection while templates >50 kb still remain.

ESTIMATED RECOVERY YIELDS FOR HIFI LIBRARY CONSTRUCTION

WORKFLOW STEPS

WORKFLOW STEPESTIMATED

YIELD

Average Post Shearing Yield (from starting gDNA) ~70 %

Average SMRTbell Yield (from sheared gDNA input into Remove ssDNA Overhangs step)

~50 %

Average Yield of ~15 kb SageELF Size-Selected Fraction (from input DNA into SageELF System)

~≥10 %*

Average yield of >15 kb BluePippin Size-Selected Fraction (from input DNA into BluePippin System)

~10 %*

* HiFi library yield after size selection is highly dependent on the final SMRTbell library size distribution

HiFi Library Sequencing Workflow Details

SAMPLE SETUP RECOMMENDATIONS FOR HIFI SMRTBELL

EXPRESS TPK 2.0 LIBRARIES – SEQUEL II SYSTEM (CHEMISTRY 2.0)

- Follow SMRT Link Sample Setup instructions using the

recommendations provided in the Quick Reference Card –

Loading and Pre-Extension Time Recommendations for

the Sequel II System*

* Sequence each size-selected fraction of interest individually

HiFi Sequencing Performance Example

Data

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1:SAGEELF SIZE-SELECTION LIBRARY RECOVERY YIELDS FOR HUMAN

GENOMIC DNA SAMPLE SHEARED TO 15 KB MODE SIZE

Pre-Size Selection

DNA Input Into

Shearing

DNA Input Into

Library Construction

DNA Input

Into SageELF

SageELF Fraction

Average Size

Size-Selected

Library Recovery

Amount (%)

9600 ng 9153 ng 3720 ng Frac 3 – 22.4 kb 568 ng (~15%)

Frac 4 – 16.6 kb 696 ng (~19%)

Frac 5 – 16.0 kb 586 ng (~16%)

Frac 6 – 12.1 kb 378 ng (~10%)

Frac 7 – 11.3 kb 240 ng (~6%)

15 kb ModeFraction of interest

(Frac 4 = 16.8 kb Mode)

Post-Size Selection

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1 (CONT.):PRIMARY SEQUENCING METRICS PERFORMANCE

Sample Name StatusMovie Time

(hours)

Pre-

extension

Time

(hours)

Total Bases

(Gb)

Unique

Molecular

Yield (Gb)

Read Length Productivity

Polymerase Longest SubreadP0 P1 P2

Mean N50 Mean N50

1Frac_4 15 kb HiFi Library

Complete 30 2 392.68 56.88 91960 181775 14514 15649 44.6% 53.3% 2.1%

HiFi data shown were generated using Sequel II Chemistry 2.0EA

15 kb Human HiFi Express 2.0 Library Achieved ~400 Gb Total Bases and >90 kb

Polymerase Read Length

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 1 (CONT.):HIFI (CCS) BASE YIELD PERFORMANCE

HiFi data shown were generated using Sequel II Chemistry 2.0EA

15 kb Human HiFi Express 2.0 Library Generated 1.8 Million ≥Q20 CCS Reads and

27 Gb of ≥Q20 CCS Bases

CCS ANALYSIS METRIC 15 KB HUMAN HIFI LIBRARY

HiFi (≥Q20) Reads 1,846,154

HiFi (≥Q20) Base Yield 27.3 Gb

HiFi (≥Q20) Read Length (Mean) 14,773 bp

HiFi (≥Q20) Read Quality (Median) Q32

Read Quality DistributionRead Length Distribution

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2:SAGEELF SIZE SELECTION LIBRARY RECOVERY YIELDS FOR E. COLI

GENOMIC DNA SAMPLE SHEARED TO 21 KB MODE SIZE

Pre-Size Selection

DNA Input Into

Shearing

DNA Input Into

Library Construction

DNA Input

Into SageELF

SageELF Fraction

Mode Size

Size-Selected

Library Recovery

Amount (%)

12,000 ng 10,000 ng 2900 ng Frac 1 – 28.8 kb 27.8 ng (~1%)

Frac 2 – 25.8 kb 904 ng (~31%)

Frac 3 – 19.4 kb 426 ng (~15%)

Frac 4 – 15.3 kb 115 ng (~4%)

Frac 5 – 13.2 kb 72.4 ng (~3%)

Mode = 21 kb Fraction of interest

(Frac 2 Mode = 25.8 kb)

Post-Size Selection

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2 (CONT.):PRIMARY SEQUENCING METRICS PERFORMANCE

Sample Name StatusMovie Time

(hours)

Pre-

extension

Time

(hours)

Total Bases

(Gb)

Unique

Molecular

Yield (Gb)

Read Length Productivity

Polymerase Longest SubreadP0 P1 P2

Mean N50 Mean N50

1Frac_2 25 kb HiFi Library

Complete 30 4 415.3 114.4 87797 169050 24791 29168 39.2 59.1 1.8

2Frac_3 20 kb HiFi Library

Complete 30 4 447.8 87.3 98645 180352 19929 21108 41.9 56.7 1.5

HiFi data shown were generated using Sequel II Chemistry 2.0

≥20 kb E. coli HiFi Express 2.0 Libraries Achieved >400 Gb Total Bases and >85 kb

Polymerase Read Length

50 kb

20 kb

25 kb HiFi Library20 kb HiFi Library

50 kb

20 kb

HIFI SMRTBELL EXPRESS TPK 2.0 LIBRARY EXAMPLE 2 (CONT.):HIFI (CCS) BASE YIELD PERFORMANCE

HiFi data shown were generated using Sequel II Chemistry 2.0

>20 kb Human HiFi Express 2.0 Libraries Generated 1.1 – 1.8 Million ≥Q20 CCS

Reads and 30 – 36 Gb of ≥Q20 CCS Bases

CCS ANALYSIS METRIC 20 KB HUMAN HIFI LIBRARY 25 KB HUMAN HIFI LIBRARY

HiFi (≥Q20) Reads 1,836,137 1,129,580

HiFi (≥Q20) Base Yield 36 Gb 30 Gb

HiFi (≥Q20) Read Length (Mean) 19,683 bp 26,149 bp

HiFi (≥Q20) Read Quality (Median) Q29 Q25

Read Quality DistributionRead Length Distribution

20 kb HiFi Library

Read Quality DistributionRead Length Distribution

25 kb HiFi Library

HiFi Sequencing Data Analysis

Recommendations for Variant Detection

and de novo Assembly Applications

HIFI SEQUENCING DATA ANALYSIS RECOMMENDATIONS FOR

VARIANT DETECTION APPLICATIONS

- Utilize SMRT Link to generate highly accurate (≥Q20) single-molecule reads (HiFi reads)

using the Circular Consensus Sequencing (CCS) analysis application

- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with

downstream analysis tools

- Can use third-party software for variant detection analysis (SNVs, InDels and SVs) using HiFi

reads generated via CCS Sequencing Mode:

- GATK

- Google Deep Variant

- Note: For structural variation detection analysis (SVs ≥20 bp) using standard Long Reads

generated via CLR Sequencing Mode, can use Structural Variant Calling analysis application

in SMRT Link or pbsv command line tools.

- Contact PacBio Technical Support (support@pacb.com) or your local Bioinformatics Field

Applications Scientist for additional information about data analysis recommendations

HiFi reads are compatible with third-party variant calling tools

HIFI SEQUENCING DATA ANALYSIS RECOMMENDATIONS FOR DE

NOVO ASSEMBLY APPLICATIONS

- Utilize SMRT Link to generate highly accurate (≥Q20) single-molecule reads (HiFi reads)

using the Circular Consensus Sequencing (CCS) analysis application

- Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with

downstream analysis tools

- Can use PacBio DevNet Tools or other third-party software for de novo assembly analysis

using HiFi reads generated via CCS Sequencing Mode:

- FALCON / FALCON-Unzip

- Hifiasm

- HiCanu

- Peregrine

- Contact PacBio Technical Support (support@pacb.com) or your local Bioinformatics Field

Applications Scientist for additional information about data analysis recommendations

HiFi reads are compatible with third-party de novo assembly tools

Technical Documentation & Applications

Support Resources

BEST PRACTICES: VARIANT DETECTION USING WHOLE GENOME

SEQUENCING WITH HIFI READS (SEQUEL II CHEMISTRY 2.0)

SMRTbell Template Preparation

- Start with unamplified genomic DNA input (≥15 µg)

- Prepare a HiFi library for using SMRTbell Express Template Prep Kit 2.0

- Enrich for 15 – 20 kb inserts with size selection

Sequence on the Sequel II System (CCS Sequencing Mode)

- Maximize output and turn-around-time with adjustable sequencing parameters

- Sequence to desired coverage based on study needs:*

- Recommend 2 SMRT Cells 8M to achieve 15-fold coverage of a human genome for comprehensive

variant detection for $2600†

Data Analysis Solutions with the PacBio Analytical Portfolio

- Detect all variant types – including SNVs, indels, SVs, and CNVs – with the highest precision

and recall using SMRT Analysis, and GATK or Google DeepVariant

- Google DeepVariant achieves higher precision and recall than GATK, particularly for indels

- Expand variant calling into previously inaccessible regions of the genome, including

repetitive regions and medically relevant genes that are difficult to map

- Output data in standard file formats – BAM and VCF – for seamless integration with

downstream analysis tools

- Phase small variants into phase blocks using WhatsHap

- Confirm variant calls visually with IGV10 and GenomeRibbon

* Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.† Prices, listed in USD, are approximate and may vary by region. Pricing includes library and sequencing reagents run on a Sequel II System and does not include instrument

amortization or other reagents.

BEST PRACTICES: WHOLE GENOME SEQUENCING (WGS) FOR

DE NOVO ASSEMBLY (SEQUEL II CHEMISTRY 2.0)

-HiFi reads provide a good balance of

read length (up to 25 kb) and accuracy

(≥99%)

- Long reads to span small repeats

- High accuracy to see minute differences

between long repeats

- Small file sizes and fast analysis time

- Lower coverage requirements for high-

quality results

- Contig N50

- Accuracy

- Genes in frame

- Phasing

- Assembly size

- Gene completeness

(BUSCO)

Contiguity

Completeness

Correctness

BEST PRACTICES: WHOLE GENOME SEQUENCING (WGS) FOR

DE NOVO ASSEMBLY (SEQUEL II CHEMISTRY 2.0) (CONT.)

- Start with >15 µg DNA

~40 kb

- Use HiFi Express

Template Prep Kit 2.0

- Size select at either >15

kb or >20 kb

- Sequence in Circular

Consensus Sequencing

(CCS) mode

- Sequence to 10-fold

coverage per haplotype

for phased assembly

- Generate HiFi reads

with CCS analysis

(command line or SMRT

Link)

- Assemble and phase

HiFi reads with

FALCON & FALCON-

Unzip or wide array of

community tools

1 2 3

TECHNICAL DOCUMENTATION & APPLICATIONS SUPPORT

RESOURCES FOR HIFI SEQUENCING

- Sequel II System Operations Guide (PN 101-774-700)

- Quick Reference Card – SMRT Link v8.0 Migration (PN 101-850-100)

- Sequel II System 8.0 Release Notes (PN 101-774-400)

- SMRT Link v8.0 Software Download Site: https://www.pacb.com/support/software-downloads/

- SMRT Link v8.0 Software Installation Instructions (PN 100-749-900)

- SMRT Link v8.0 Release Notes (PN 100-749-600)

- SMRT Link v8.0 User Guide (PN 101-039-100)

- SMRT Tools Reference Guide (v8.0) (PN 100-939-900)

- Sequel SMRT Link Web Services API Use Cases (v8.0) (PN 101-430-800)

- Quick Reference Card – Loading and Pre-Extension Recommendations for the Sequel II System (PN 101-

769-100)

- Pacific Biosciences Glossary of Terms (PN 000-710-267)

Sequel II System and SMRT Link Documentation

https://www.pacb.com/support/documentation/

TECHNICAL DOCUMENTATION & APPLICATIONS SUPPORT

RESOURCES FOR HIFI SEQUENCING (CONT.)

- Procedure & Checklist – Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit

2.0 (PN 101-853-100)

- Procedure & Checklist - Preparing SMRTbell Libraries Using Express Template Prep Kit 2.0 With Low

DNA Input (PN 101-730-400)

- Procedure & Checklist – Using AMPure PB Beads for Size-Selection (PN 101-854-900)

- Application Brief: Variant Detection using Whole Genome Sequencing With HiFi Reads – Best Practices

(PN BP106-092419)

- Application Brief: Whole Genome Sequencing (WGS) for De novo Assembly – Best Practices (PN

BP102-121219)

- Overview – Sequel Systems Application Options and Sequencing Recommendations (PN 101-851-300)

https://www.pacb.com/support/documentation/

Sample Library Preparation Protocol Documentation & Application Briefs

Appendix I: Optional Alternative Shearing

Method Using Covaris g-TUBEs

DNA SHEARING USING COVARIS G-TUBES

Specific guidance for shearing gDNA to ≥15 – 20 kb using the Covaris g-TUBE

(modified from the Covaris User Manual) is provided below and should be strictly

followed to achieve the desired target fragment size.

1. Dilute 15 µg high molecular weight gDNA in 1X Elution Buffer to a concentration of 83.3

ng/µL in a volume of 180 µL.

2. Transfer gDNA to g-TUBE and shear the gDNA at 2029 x g (5500 rpm in the Eppendorf

MiniSpin Plus) for 2 minutes.

3. Check for any residual sample remaining in the upper chamber. If present, re-spin for

another 2 minutes. Repeat spin until entire gDNA sample has passed through the orifice.

4. Invert and spin 2029 x g (5500 rpm in the Eppendorf MiniSpin Plus) until entire gDNA

sample has passed through the orifice.

5. Repeat step 4, FOUR times for a total of SIX passes through the orifice.

6. Transfer the sheared gDNA to a new 2.0 mL DNA Lo-bind microfuge tube.

7. Proceed to the “Concentrate DNA using AMPure PB Beads” section in the Procedure &

Checklist to concentrate your sheared gDNA using AMPure beads.

SIZING QC OF GENOMIC DNA SHEARED USING g-TUBES

- Femto Pulse sizing QC of sheared gDNA shows a mode of ~20 kb

Appendix II: Optional Alternative Size-

selection Method Using the Sage Science

BluePippin System for Variant Detection

Applications

SIZE SELECTION WITH THE BLUEPIPPIN SYSTEM

- BluePippin size selection protocol options for HiFi library construction:

- Option 1: Size-select a single fraction (>15 kb) – Recommended size-selection method for de

novo assembly applications [see Slides 41 – 44 in this presentation for description of procedure]

- Option 2: Size-select two fractions (9 – 13 kb and >15 kb) – Can be used to size-select HiFi

libraries for variant detection applications if a Sage Science SageELF system is unavailable [see

Slides 67 – 71 in Appendix II of this presentation for description of procedure]

- For the latest BluePippin User Manual and guidance on size-selection protocols, please

contact Sage Science (www.sagescience.com)

- Note: Visit Sage's website (http://www.sagescience.com) to verify that your BluePippin

software is up-to-date.

As an alternative to the SageELF system, Sage Science’s BluePippin tool may also

be used to size-select libraries for HiFi sequencing

OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT

DETECTION APPLICATIONS

1. Prepare up to 5 µg of SMRTbell templates in a final volume of 30 μL Elution Buffer for

each Blue Pippin lane.

2. Bring the Loading Solution to room temperature, and then add 10 μL of the Loading

Solution to the 30 μL DNA sample. For loading multiple lanes with the same sample, scale

the volumes proportionally. The Loading Solution is viscous, so pipet slowly to ensure

complete transfer into the DNA sample.

a) Pipette mix using wide-bore pipette tips to mix.

b) Spin briefly to collect the contents at the bottom of the tube.

3. Follow the manufacturer’s recommendations to set up a run protocol.

a) Select the “0.75% DF Marker S1 3-10 kb Improved Recovery” Cassette Definition File for your

sample.

b) Using the “Range” selection mode, enter a desired “BPstart” value of 9000 and a BP End value of

13000.

Note: Sample lanes containing <3 µg of SMRTbell library material will run faster during

electrophoresis. In such cases, PacBio recommends adjusting the BP Start value to 8000 bp and

BP End value to 12000 bp. We do not recommend running lanes with <2 µg of SMRTbell library

material.

c. Be sure to assign a marker lane. We recommend using Lane 4 for the marker.

OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT

DETECTION APPLICATIONS (CONT.)

4. Load the S1 marker and samples into the BluePippin gel cassette and start the run. Run

time is approximately 4.5 hours.

5. Collect the 9-13 kb fraction: To maximize recovery of eluted DNA, wait at least 20

minutes after the run terminates before removing the sample from the elution chamber.

a. Collect the eluate containing the 9-13 kb fraction into a 2.0 mL DNA LoBind tube.

b. Wash the elution well with 40 µL of Sage Science’s Electrophoresis Buffer and add the recovered

wash liquid to the eluted sample. A second wash may further increase recovery yields.

c. Refill the elution chamber with 40 µL Electrophoresis Buffer and re-seal the chamber. Close the lid

of the BluePippin.

6. Collect the ≥15 kb fraction: Use Manual Mode to collect a second fraction.

a) From the Main Tab, select “Manual Mode” from the top of the “Protocol Name” drop down menu.

b. Click “Start” on the controller.

c. Click “Elute” box for each sample lane. Box will turn orange and current will be

detected.

d. When elution timer reaches 30 minutes, click “Idle” box for each sample lane. Idle box will become

white and current will drop.

e. Collect second eluate as described in Step 5 above, including washes. Typical insert size for this

manually eluted fraction is ~15kb.

7. Proceed to the final 0.5X AMPure PB Bead purification step in the Procedure & Checklist,

or store the collected fractions at -20°C for future use.

OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT

DETECTION APPLICATIONS (CONT.)

OPTIONAL ALTERNATIVE BLUEPIPPIN SIZE SELECTION PROTOCOL: SIZE-SELECT TWO FRACTIONS (9 – 13 KB AND >15 KB) FOR VARIANT

DETECTION APPLICATIONS (CONT.)

COLLECTED

FRACTION

MEAN SIZE

(KB)

1 12.2

2 19.2

BP Fraction 1(Mode = 12.6 kb)

BP Fraction 2(Mode = 17.7 kb)

Example Femto Pulse Sizing QC for 12 kb & 19 kb BluePippin Size-Selected HiFi

Library Fractions

Femto Pulse sizing QC analysis for a human library sample size-selected on the BluePippin system using the “0.75% DF Marker

S1 3-10 kb Improved Recovery” cassette definition file to collect two size fractions (9 – 13 kb and >15 kb). Femto Pulse smear

analyses show that that the mean SMRTbell library size for Fraction 1 is 12.2 kb and the mean size for Fraction 2 is 19.2 kb.

Appendix III: Optional Alternative Size-

selection Method Using AMPure PB Size-

Selection for De Novo Assembly

Applications

- Procedure & Checklist (PN 101-854-900) provides instructions

for using AMPure PB beads for size-selection of SMRTbell

libraries

- Note: AMPure PB Bead size-selection is not the primary

recommendation for HiFi de novo assembly applications since

it will result in shorter HiFi read lengths compared to using a

BluePippin system. AMPure PB Bead size-selection may,

however, be a useful option when sample quantity is limited.

- AMPure PB beads are diluted with PacBio Elution Buffer to

achieve a specified volumetric ratio and then used for size-

selection

- Important: To efficiently remove SMRTbell templates <3 kb or

<5 kb, be sure to use this procedure after the first AMPure PB

bead purification step (post-adapter ligation) in the HiFi

SMRTbell library construction workflow.

- Protocol document contains:

1. List of required materials

2. Procedure for diluting AMPure PB Beads in Elution Buffer

to achieve a specified volumetric concentration

3. Procedure for performing size selection to remove <3 kb

SMRTbell templates or <5 kb SMRTbell templates using

a specified volumetric ratio of diluted AMPure PB

Beads:Sample

PROCEDURE & CHECKLIST – USING AMPURE PB BEADS FOR SIZE-

SELECTION

Optional alternative AMPure PB size-selection protocol for use with HiFi de novo

assembly applications

https://www.pacb.com/support/documentation/

LIST OF REQUIRED MATERIALS AND EQUIPMENT FOR AMPURE PB

SIZE SELECTION PROCEDURE

To help minimize shearing-induced damage to SMRTbell templates, use wide-bore

pipette tips for pipette mixing AMPure PB Bead/DNA solutions

DILUTION OF AMPURE PB BEADS WITH ELUTION BUFFER

The final AMPure PB bead concentration is critical to the success of this procedure.

Therefore, accurate pipetting is of utmost importance to achieve a final 35% (v/v)

AMPure PB Bead working solution in Elution Buffer

SIZE-SELECTION PROCEDURE USING DILUTED AMPURE PB BEADS

Key Considerations

- Important: To efficiently remove SMRTbell templates <3 kb or <5 kb from your HiFi library

sample, be sure to use this procedure after the first AMPure PB bead purification step

(post-adapter ligation) in the SMRTbell library construction workflow.

- For effective size-selection using AMPure PB beads, accurate pipetting is necessary.

- Additionally, the DNA concentration of the SMRTbell library to be size-selected must be

0.5–10 ng/μL.

- Use of higher concentrations (>15-100 ng/μL) decreases the efficiency of reduction of short insert

SMRTbell templates. Adjust the sample concentration so that the DNA concentration is within this

range.

- Note: that presence of ethanol in the sample impacts yield - therefore it is important to

remove residual ethanol in the library prior to performing the AMPure PB size selection

procedure

- The volume of diluted AMPure PB beads to use for size-selection depends on the desired

molecular weight cutoff (see next slide).

- To remove <3 kb SMRTbell templates, use 3.7X ratio of diluted AMPure PB beads:sample.

- To remove <5 kb SMRTbell templates, use 3.1X ratio of diluted AMPure PB beads:sample.

- Note: It is critical to mix precise volumes of the sample and diluted AMPure PB beads to achieve

successful size-selection.

- SMRTbell library recovery yields with AMPure PB size-selection are typically >40%

SIZE-SELECTION PROCEDURE USING DILUTED AMPURE PB BEADS

(CONT.)

AMPure PB Size Selection Performance Is Dependent on the Volumetric Ratio of

Diluted AMPure PB Beads:Sample Used

- PacBio recommends using a 3.7X ratio of diluted AMPure PB Beads:Sample to remove

<3 kb SMRTbell templates and a 3.1X ratio to remove <5 kb SMRTbell templates.

Diluted AMPure PB Bead:Sample Volumetric Ratio

Siz

e (

bp

)

Figure shows the performance of diluted (35% v/v) AMPure PB beads for size-selection of a DNA sample

with a concentration of 8 ng/μL at varying ratios of diluted AMPure PB bead Volume:Sample Volume.

HIFI DE NOVO ASSEMBLY EXAMPLE USING AMPURE PB SIZE

SELECTION: DROSOPHILA GENOME ASSEMBLY

SampleSageELF

19 kb Fraction

SageELF

24 kb FractionAMPure PB SS

AMPure PB SS+15 kb In silico Cut

HiFi Base Yield

(Coverage)

31 Gb

(221-fold)

26 Gb

(186-fold)

33 Gb

(236-fold)

28 Gb

(200-fold)

Assembly Coverage

(Down-sampled)20-fold 20-fold 20-fold 20-fold

Assembly Length 156 Mb 160 Mb 165 Mb 163 Mb

Contig N50 6.46 Mb 9.30 Mb 4.27 Mb 5.71 Mb

N Contigs 332 278 349 297

AMPure PB size-selected HiFi library generated Drosophila genome assemblies

comparable in quality to SageELF size-selected HiFi libraries

General Recommendations for High-

Molecular Weight gDNA Isolation, QC &

Handling for SMRTbell Library Construction

Human

Biomedical

Research

Plant & Animal

Sciences

Microbiology

& Infectious

Disease

- If gel purification is required, avoid using ethidium/UV based visualization methods. One

alternative is to use SYBR® Safe (Invitrogen) and visualize with blue light

- To help resuspend the DNA, carefully invert the tube several times after adding buffer

and/or tap the tube gently. Avoid vortexing genomic DNA when possible as vortexing can

cause shearing of the DNA. It is also recommended to use wide bore tips in sample

handling

- Alternatively, allow the DNA to stand in buffer overnight at 25°C to resuspend

- Overheating can introduce DNA damage. Inactivate DNAase as recommended by the

vendor kit. It is best to avoid heat inactivation when possible

- DNA storage conditions: 4 °C (short-term); -20°C / -80°C (long-term)

GENERAL RECOMMENDATIONS FOR ISOLATING HIGH-MOLECULAR

WEIGHT GENOMIC DNA

EXAMPLE THIRD-PARTY HIGH-MOLECULAR WEIGHT GENOMIC

DNA ISOLATION PROTOCOL AND KIT SOLUTIONS

Note: The products below have not been extensively tested or validated by PacBio

R&D but are listed here as examples of third-party kits or methods used by other

PacBio customers for isolating genomic DNA for SMRTbell library preparation

Plant Tissue

QIAGEN Genomic-tip 20/100/500/G Kit

Unsupported Protocol – Switchgrass (Panicum virgatum) DNA isolation [USDA]

- http://www.pacb.com/wp-content/uploads/2015/09/Switchgrass-DNA-isolation.pdf

Unsupported Protocol – DNA extraction of Chlamydomonas using CTAB [JGI]

- http://www.pacb.com/wp-content/uploads/2015/09/DNA-extraction-chlamy-CTAB-JGI.pdf

QIAGEN User-Developed Prototocol: Isolation of genomic DNA from plants and filamentous fungi

using the QIAGEN Genomic-tip Kit

- https://www.qiagen.com/de/resources/resourcedetail?id=cb2ac658-8d66-43f0-968e-

7bb0ea2c402a&lang=en

Circulomics Nanobind Plant Nuclei Big DNA Kit

- https://www.circulomics.com/

Insect Tissue

QIAGEN User-Developed Prototocol: Isolation of genomic DNA from mosquitoes or other insects using

the QIAGEN Genomic-tip Kit

- https://www.qiagen.com/ca/resources/resourcedetail?id=b45c3cc3-7f2b-4f4a-aa37-

21d814ed3730&lang=en

Circulomics Nanobind Tissue Big DNA Kit

- https://www.circulomics.com/

Fish Tissue

QIAGEN Genomic-tip 20/100/500/G Kit

- Reference: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered

Scaffolding. PLOS Genetics 12(4): e1005954. (2016) doi: 10.1371/journal.pgen.1005954

http://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1005954

QIAGEN User-Developed Prototocol: Purification of archive-quality DNA from 10–20 mg fish tissue

using the Gentra® Puregene® Tissue Kit or Gentra Puregene Mouse Tail Kit

- https://www.qiagen.com/ca/resources/resourcedetail?id=948e5a5e-57c4-482f-a852-

43ec3da97fce&lang=en

Circulomics Nanobind Tissue Big DNA Kit

- https://www.circulomics.com/

Human Tissue

QIAGEN Genomic-tip 20/100/500/G Kit / QIAGEN Gentra Puregene Cell Kit

Unsupported Protocol – Gentra Puregene Cell Kit (Qiagen) DNA Isolation [Univ. Washington]

- http://www.pacb.com/wp-content/uploads/2015/09/Gentra-Puregene-Qiagen-DNA-Isolation.pdf

Reference: Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608-611 (29

January 2015) doi:10.1038/nature13907 http://www.nature.com/nature/journal/v517/n7536/full/nature13907.html

Circulomics Nanobind CBB Big DNA Kit or Nanobind Tissue Big DNA Kit

- https://www.circulomics.com/

Macherey-Nagel™ NucleoBond™ AXG 20/100/500 Gravity-flow Columns

- https://www.mn-net.com/

NEW SAMPLE PREPARATION ONLINE RESOURCE

Literature resource for sample collection and DNA extraction protocol references

- PacBio does not assume responsibilities/guarantees for these external publications/protocols, but we are

happy to help as best as we can to guide/connect. Please contact ExtractDNA@pacb.com for more

discussions around your particular species & sequencing project!

www.ExtractDNAforPacBio.com

High Molecular Weight gDNA and SMRTbell Library Cleanup

AMPure PB Bead Wash

- Refer to the appropriate Procedure & Checklist protocol document for specific recommendations for AMPure PB bead

purification of different SMRTbell library insert sizes

GENERAL RECOMMENDATIONS TO HELP CLEAN UP HIGH

MOLECULAR WEIGHT GENOMIC DNA AND SMRTBELL LIBRARIES

Starting with high-quality, high molecular weight (HMW) genomic DNA will result

in longer libraries and better de novo assembly performance

METHODS FOR EVALUATION OF GENOMIC DNA QUALITY

- Input genomic DNA must be carefully QC’d to assess integrity

- PFGE/FIGE or Femto Pulse sizing tool is highly recommended

- High molecular weight DNA → long read lengths

- Degraded DNA → short read lengths, low library recovery yields (dependent on size selection

parameters)

- DNA purity can be determined by using a NanoDrop instrument or other spectrophotometer device

- PacBio highly recommends using the Qubit High Sensitivity fluorometric assay for accurate dsDNA

quantitation

1.CHEF Mapper XA System (Bio-Rad)

2.Femto Pulse System (Agilent)

3.Pippin Pulse System (Sage Science)

A. DNA Sizing Characterization

http://www.sagescience.com/products/pippin-pulse/

http://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems

https://www.agilent.com/en/product/automated-electrophoresis/femto-pulse-

systems

Recommended methods for determining gDNA size distribution:

Lane 1: 8-48 kb Ladder (Bio-Rad)

Lane 2: 5 kb Ladder (Bio-Rad)

Lane 3: HMW gDNA

Lane 4: Degraded gDNA

Lane 1: High MW gDNA

Lane 2: Degraded gDNA

Lane 3: 165 kb Ladder

Evaluation of gDNA quality using A) Bio-Rad CHEF

Mapper System and B) Femto Pulse System. Lanes 3 A and

1B are examples of high quality, high molecular weight

genomic DNA. Lanes 4A and 2B are examples of degraded

gDNA.

A B➢ Up to 10 Mb

➢ >16 Hour Run Time

➢ Up to 165 kb

➢ 1.5 Hour Run Time

➢ Up to 80 kb

➢ 16 Hour Run Time

Femto Pulse System only requires

~0.5 ng of gDNA sample and

assay run time is <1.5 hours

- DNA purity can be determined by using a NanoDrop® instrument or other spectrophotometers

- For ultrapure gDNA, A260/280 ratio is typically between ~1.8 - 2.0 and A260/230 ratio is ≥2.0

- If A260/280 and A260/230 readings are out of the range specified above, PacBio recommends

performing an AMPure® purification step followed by re-assessment of quantity and purity of

the gDNA sample

260/280 Ratio

▪ A low A260/A280 ratio may indicate the presence of protein, phenol, or other contaminants that absorb

strongly at or near 280 nm. Sometimes it may be caused by a very low concentration of nucleic acid.

▪ High 260/280 ratios are not indicative of an issue

260/230 Ratio

▪ A low A260/A230 ratio may be the result of:

❑ Carbohydrate carryover (often a problem with plants)

❑ Residual phenol from nucleic acid extraction

❑ Residual guanidine (often used in column-based kits)

❑ Glycogen used for precipitation

▪ A high A260/A230 ratio may be the result of:

❑ Making a blank measurement on a dirty pedestal of a Nanodrop instrument

❑ Using an inappropriate solution for the blank measurement

B. DNA Purity Determination

- Accurate quantitation of DNA concentration is critical for PacBio template preparation

procedures.

▪ Specifically, it is critical to determine the concentration of the double-stranded DNA, since only double-

stranded DNA will be converted into sequencing templates.

- PacBio highly recommends using a Qubit® fluorometer tool and Qubit ds DNA High

Sensitivity (HS) Assay Kit for routine DNA quantitation during SMRTbell library construction.

-When assessing gDNA QC, PacBio recommends using both fluorometric and

spectrophotometric methods – for example, using both the Qubit and NanoDrop instruments

▪ If the sample is pure gDNA, free of any RNA contaminants and other small molecules, the two methods

should converge to similar DNA concentration measurement values

C. DNA Quantification

- If the measured NanoDrop concentration is significantly different

(>50%) from the Qubit measurement, PacBio recommends doing an

AMPure purification step (as specified by your chosen library

preparation protocol), followed by a re-measurement with both

methods. Typically, a single AMPure purification step resolves the

discrepancy.

▪ If the concentration measurement discrepancy after one AMPure purification

step is not reduced, we recommend trying another cleanup approach before

a re-measurement with both methods.

SPECIAL HANDLING RECOMMENDATIONS FOR

PREPARING LARGE INSERT (>15 KB)

SMRTBELL LIBRARIES

1. For small numbers of samples, use DNA Lo-Bind 1.5 mL microcentrifuge tubes for all

enzymatic and AMPure PB bead purification steps.

2. Multi-channel pipettes and 96-well plates/PCR strip tubes may be used to efficiently

process large numbers of samples.

3. Use wide-bore tips for all pipette mixing steps when preparing >15 kb size-selected

libraries.

4. Never vortex tubes containing high-molecular weight DNA samples. Mix by gentle pipetting

unless stated otherwise.

5. Always follow best practices for DNA quantitation using a Qubit fluorometer system:

- Use the Qubit dsDNA High Sensitivity (HS) Assay reagent kit.

www.pacb.com

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo, PacBio,

SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method. Use of this

method may require rights to third-party owned intellectual property. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. FEMTO Pulse and

Fragment Analyzer are trademarks of Agilent Technologies Inc.

All other trademarks are the sole property of their respective owners.