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Amplicon sequencing using next-‐gen technology
STAMPS course 2013 Hilary Morrison
& Sharon Grim, Joe Vineis
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Overview • Tag sequencing • Primer design • Amplicon sequencing on two Illumina plaIorms – fusion primers – opKmizaKon – piIalls – current protocols
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WHAT YOU WILL NOT HEAR ABOUT
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RETIRED
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EXCHANGED
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Targets
• Ribosomal RNA (SSU, LSU) • Internal transcribed spacers (ITS)—fungi • FuncKonal genes – nirS – butyrate kinase, butyrate transferase – others
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Which variable regions to target?
V1 V3 V7 V8 V9
8F
V2 V4 V5 V6
V6 (967F-‐1046R) 60 bp V4 (518F-‐806R) 288 bp V4V5 (518F-‐1064R) 550 bp V9 (1380/1389-‐1513) for eukaryotes
341 518 806 926 967 1064 1513 1380
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ITS (fungi)
18S
ITS1 ~350-‐400 bp ITS2 ~250-‐300 ITS1-‐ITS2 ~550-‐800
ITS1F ITS1R/ITS2F ITS2R
ITS1 ITS2 28S 5.8S
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AmplificaKon process • DOs:
– High quality DNA template: e.g. 260/280 ~2 – Replicate reacKons using high-‐fidelity polymerase – NegaKve controls – Pool, clean, size-‐select
• Qiagen MinElute (FLX; Illumina v6), • Ampure (Titanium; Illumina v4v5); • Pippin Prep (Illumina pools)
– QuanKtaKon • e.g. PicoGreen, qPCR
– VisualizaKon • Bioanalyzer, Caliper, gel
• DON’Ts: – MDA or nested amplificaKon
Pre-‐Ampure Post-‐Ampure
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What can go wrong? (sources of error) • ContaminaKon • PCR error (enzyme misincorporaKon) • Chimeras • PCR ectopic amplificaKon (non-‐rRNA) • MulKple Sequence Alignment • Distance measurements (gaps, etc.) • Clustering methods
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Post sequencing • Basecalling • QC pipeline • Taxonomy assignment or clustering – QIIME – GAST (Sue Huse, 8/4) – mothur – RDP Pipeline (hjp://rdp.cme.msu.edu/) – Oligotyping – basic BLAST
• VAMPS
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16S-‐SPECIFIC PRIMER DESIGN
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Design primer suite for conserved sites
• Start with known primers • Check matches against SILVA or other rRNA database – Phyla missed? – ProporKon missed?
• Add variants or degenerate sites to expand coverage
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Literature review
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Database sources
3,194,778 Total 739,633 SSURef
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Example: bacterial-‐archaeal universal primer v. 1
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
0 mismatches 1 mismatch 2 mismatches 3 mismatches
Bacteria
Archaea
Eukarya
GGACTAC[ACG][CG]GGGTATCTAAT
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Example: bacterial-‐archaeal universal primer v.2
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
0 mismatches 1 mismatch 2 mismatches 3 mismatches
Bacteria
Archaea
Eukarya
GGACTAC[ACG][CG]GGGTATCTAAT GGACTAC[ACT][ACG]GGGT[AT]TCTAAT
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
0 mismatches 1 mismatch 2 mismatches 3 mismatches
Bacteria
Archaea
Eukarya
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V6 primers 967F is pool of 4 oligos with 2-‐16X degeneracy
CNACGCGAAGAACCTTANC CAACGCGMARAACCTTACC ATACGCGARGAACCTTACC CTAACCGANGAACCTYACC
1046R: 4 oligos or 1 oligo with 16X degeneracy CGACAGCCATGCANCACCT CGACAACCATGCANCACCT CGACGGCCATGCANCACCT CGACGACCATGCANCACCT
CGACRRCCATGCANCACCT
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V6 Primer Sequence Target RefDB exact match
967F1-‐PP CNACGCGAAGAACCTTANC Bacteria 63.3%
967F-‐AQ CTAACCGANGAACCTYACC Deferribacteres, Aquificales 0.1%
967F-‐U12 CAACGCGMARAACCTTACC AddiKonal Proteobacteria 14.0%
967F-‐U3 ATACGCGARGAACCTTACC AddiKonal Bacteroides; Spirochaetes 12.7%
1046R CGACRRCCATGCANCACCT Bacteria 95.4%
Keep level of degeneracy low (2-‐16X) Are rare, but important groups missed? Primers with 1-‐2 mismatches open work—pay ajenKon to Tm
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V4V5 Primer Sequence Target RefDB exact match
518F CCAGCAGCYGCGGTAAN Bacteria 97.0%
926R1 CCGTCAATTCNTTTRAGT Bacteria 96.2%
926R3 CCGTCAATTTCTTTGAGT Verrucomicrobia,
Delta proteobacteria, AcKnobacteria
2.3%
926R4 CCGTCTATTCCTTTGANT Epsilon proteobacteria 1.3%
Why not use CCGTCWATTYNTTTRANT?
128 variants and only 40 match a known bacterial sequence!
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Fusion primer modificaKons • Phosphorothioate linkages • Barcoding: 5-‐12 nt • Indexing: 6-‐12 nt • Fusion with plaIorm-‐specific sequencing primer sequences (vs. ligaKon)
• Illumina fusion primers include bridge adapter regions
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ILLUMINA TAG SEQUENCING
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Illumina amplicon strategy
• Fused primer approach, not adapter ligaKon – Primers much longer than 454 versions – Include bridge adapter sequences – Minimize cost of barcoded primer sets
• Many labs now have Knight 515F/806R sets (V4; >1000 indices) • CombinaKon of 12 indices x 8 inline barcodes creates 96-‐plex set for other targets
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Illumina amplicon targets
• How long are the reads? – Hiseq: 100/150 PE – Miseq 250 PE and longer
• How good is the quality towards the end? – Undetected errors will inflate alpha-‐diversity – Merged reads require overlap
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100 nt PE HiSeq1000
V6R: 1046R
~60 bp complete overlap
967F
Read 1
Read 2
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Longer reads on MiSeq
V5R: 926R
50-‐100 nt overlap
V4F: 518F
Read 1
Read 2
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Start with v6
• Original 454 pyrotag target-‐lots of experience • Sequence overlap with v6v4 datasets • Cheaper to do • Greater sampling depth – 300-‐400 million clusters per lane possible – 3M reads at 96-‐plex – 300K reads at 960-‐plex
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rRNA…TAATCTWTGGGVHCATCAGG-CCGACTGACTGA-TTGCGTGCGATC-TAGAGCATACGGCAGAAGACGAAC!
5' AATGATACGGCGACCACCGAGATCTACAC-TATGGTAATTGT-GTGCCAGCMGCCGCGGTAA…rRNA!
5’ adapter “pad/linker”*
*designed to have no homology to 16s or Illumina adapters/primers
“515F”
Knight lab sequencing primer 1
“806R-‐rc” “pad/linker-‐rc”* index-‐rc 3’ adapter-‐rc
Knight lab sequencing primer 2 RC is indexing sequencing primer
Fusion primer design
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Learned during Miseq Training
• First 4 bases of read 1 used for cluster finding (no change)
• Next 8 bases (up through base 12) need to be high-‐complexity because of phasing/prephasing calculaKon
• This is also true of read 2 • Thus, need to have randomness in cycles 1-‐12 of both reads!
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In-‐line bar codes vs. indices • In-‐line bar codes cut into sequencing reads, but: • Cluster finding requires random distribuKon of A,C,G,T at
start of read 1 • We designed our primers to provide this diversity
– 5’ NNNN (1-‐4) – Careful mix of bar codes (5-‐9)
• Didn’t work – Clusters are found, but amplicons are sKll ‘low complexity’ samples
– Diversity needed at first 12 bases of both read 1 and read 2 • Had to add high proporKon of PhiX or other ‘random’
library
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5’ AATGATACGGCGACCACCGAGATCTACAC-‐ TCTTTCCCTACACGACGCTCTTCCGATCT-‐ NNNNTCAGC-‐ CNACGCGAAGAACCTTANC 3’
967F_PP_N4TCAGC: one of 4 primers with TCAGC barcode; forward read
Fusion primer design
5’ CAAGCAGAAGACGGCATACGAGAT-‐ CGTGAT-‐ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-‐ CGACRRCCATGCANCACCT 3’
1046R_index_1 : single degenerate primer with index; reverse read
Bridge adapter Sequencing primer binding In-‐line barcode 16S PCR
Bridge adapter Index Sequencing primer binding (x2) 16S PCR
Similar for v4v5 suite
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Primer synthesis • >80 nt long • PurificaKon needed – DesalKng only – PAGE (used for first set; expensive) – No other opKon because of oligo length
• Pooled 4 967F primers for each barcode (V6) • Pooled 3 926R primers for each index (V4V5) • Final set of 8 forward/barcoded and 12 reverse/indexed primers = 96 unique combinaKons for each domain
• Working stocks @10 uM each
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ILLUMINA SEQUENCING
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Effect of low-‐complexity libraries
• Clusters found correctly • Then read hits highly
conserved sequence • Quality scores reduced • MiKgate with lower cluster
density and HC library spike-‐in – over 50% PhiX on early Miseq runs
• Illumina has sopware fix for problem on Miseq – now ~2% PhiX on Miseq runs
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Control gDNA: known community
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Error rate
• Generated data from HMP mock community gDNA • Expected sequences recovered • Aligned unique sequences to reference • Sequencing errors, not quality scores
• Overall error: 0.52%
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How does Illumina tag sequencing compare to pyrotag sequencing?
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Analysis of v6 data from real samples
Sandra Mclellan samples: Illumina (HiSeq) overrepresents AcKnomyces; misses Arcobacter almost enKrely
Samples are: 3 HiSeq v6 amplicon datasets and 2 454 datasets (v6v4 and v6 only)
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Does length of primers bias recovery?
Test alternaKves in 2-‐step PCR. – 16s primers only, followed by full-‐length fusion – 16s plus part of Illumina adapters, followed by adapters to bridge
Sample, condidons Barcode Index
SS_WWTP_1_25_11, 16s only 20 cycles GAGAC 7: GATCTG/CAGATC
SS_WWTP_4_11_11, 16s only 20 cycles ACGCA 8: TCAAGT/ACTTGA
SS_WWTP_1_25_11, split primers TCAGC 1:CGTGAT
SS_WWTP_4_11_11, split primers TCAGC 2:ACATCG
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“Split” primers
967F_PP+seq CACGACGCTCTTCCGATCTACGTTCAGCCNACGCGAAGAACCTTANC
967F_AQ+seq CACGACGCTCTTCCGATCTCGTATCAGCCTAACCGANGAACCTYACC
967F_U2+seq CACGACGCTCTTCCGATCTGTACTCAGCCAACGCGMARAACCTTACC
967F_U3+seq CACGACGCTCTTCCGATCTTACGTCAGCATACGCGARGAACCTTACC
ACGTbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACGT
CGTAbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTA
GTACbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAC
TACGbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTACG
1046R+seq GGAGTTCAGACGTGTGCTCTTCCGATCTCGACRRCCATGCANCACCT
BridgeIndex_1 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCC
BridgeIndex_2 CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCC
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PCR condiKons Standard 3 x 33 ul amplicon reacKon plus negaKve control 1st round: either 16s only primers or 16s+parKal adapter Only 20 cycles (vs. 30) Pooled 3 reacKons Diluted 10 ul to 500 (1:50) Used 1 ul diluKon in 2nd round of PCR with 2nd set of primers, 5 cycles
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PCR results
16s only, 20 cycles 16s only, +5 cycles 16s only, negaKve 16s only, 20 cycles 16s only, +5 cycles 16s only, negaKve Split, 20 cycles Split, +5 cycles Split, negaKve Split, 20 cycles Split, +5 cycles Split, negaKve
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Sequencing results
Searched for match in raw data, read 1, each dataset >gi|343201492 Arcobacter cibarius TGGACTTGACATAGTAAGAACTTTCTAGAGATAGATTGGTGTCTGCTTGCAGAAACTTATATAC
Sample, condidons Count read1 Count Arcobacter %
SS_WWTP_1_25_11_Bv6v4 35,612 5,525 15.50%
SS_WWTP_1_25_11_ACGCA 214,950 2,309 1.10%
SS_WWTP_1_25_11_GACTC 240,520 2,606 1.10%
SS_WWTP_1_25_11_GTATC 392,570 5,445 1.40%
SS_WWTP_1_25_11_16s 6,106,654 900,824 14.80%
SS_WWTP_1_25_11_split 12,782,547 1,558,392 12.20%
SS_WWTP_4_11_11_16s 5,779,659 824,789 14.30%
SS_WWTP_4_11_11_split 17,102,934 2,108,534 12.30%
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0"
5"
10"
15"
20"
25"
30"
35"
40"
45"
Beijerinckiaceae"
Alicyclobacillaceae"
Helicobacteraceae"
Xiphinematobacteriaceae"
Crenotrichaceae"
Rhodobacteraceae"
Auran@monadaceae"
Acetobacteraceae"
Xanthobacteraceae"
Tracheophyta"
Erythrobacteraceae"
Enterococcaceae"
Lactobacillaceae"
Phyllobacteriaceae"
Neisseriaceae"
Coxiellaceae"
Sinobacteraceae"
Listeriaceae"
Clostridiaceae"
Chi@nophagaceae"
RickeJsiaceae"
Bacteroidaceae"
Williamsiaceae"
Staphylococcaceae"
Rhodocyclaceae"
Streptococcaceae"
Nocardiaceae"
Nocardioidaceae"
Burkholderiaceae"
Bdellovibrionaceae"
Methylophilaceae"
Family"I"
Bacillaceae"
Brucellaceae"
Cryomorphaceae"
Microbacteriaceae"
Planctomycetaceae"
Bradyrhizobiaceae"
Moraxellaceae"
Legionellaceae"
Hyphomicrobiaceae"
Methylobacteriaceae"
Unassigned"
Rhodospirillaceae"
Oxalobacteraceae"
Paenibacillaceae"
Alcaligenaceae"
Caulobacteraceae"
Verrucomicrobiaceae"
Comamonadaceae"
Xanthomonadaceae"
Sphingobacteriaceae"
Sphingomonadaceae"
Rhizobiaceae"
Enterobacteriaceae"
Pseudomonadaceae"
Cytophagaceae"
Flavobacteriaceae"
MTG$vs$v4v5$
M51.MTG.RC"
M51.MTG.UC"
M51.v4v5.1step"
M51.v4v5.2step"
0"
5"
10"
15"
20"
25"
30"
35"
Micromonosporaceae"
Xanthobacteraceae"
Streptomycetaceae"
Nitrosomonadaceae"
Hyphomonadaceae"
Pseudonocardiaceae"
Methylocystaceae"
Hydrogenophilaceae"
Micrococcaceae"
Rhodospirillaceae"
Rhodocyclaceae"
SAR116"
Beijerinckiaceae"
Unassigned"
PiscirickeFsiaceae"
Paenibacillaceae"
Patulibacteraceae"
Oxalobacteraceae"
Psychromonadaceae"
Bacillaceae"
Erythrobacteraceae"
Acetobacteraceae"
Nocardioidaceae"
Bartonellaceae"
Phyllobacteriaceae"
Hyphomicrobiaceae"
Francisellaceae"
Bradyrhizobiaceae"
Neisseriaceae"
Mycobacteriaceae"
Alteromonadaceae"
Methylobacteriaceae"
Brucellaceae"
Enterobacteriaceae"
Cytophagaceae"
Verrucomicrobiaceae"
Microbacteriaceae"
Caulobacteraceae"
Mycoplasmataceae"
Alcaligenaceae"
Rhizobiaceae"
Xanthomonadaceae"
Nocardiaceae"
Sphingomonadaceae"
Sphingobacteriaceae"
Flavobacteriaceae"
Comamonadaceae"
Moraxellaceae"
Pseudomonadaceae"
MTG$vs$v4v6$
50ng.8cyc.R2"%"
367.v4v6"%"
Other evidence for ‘missing’ taxa
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Arcobacter
Epsilons Rikenella
Primer Tm at 1.5-‐3.6mM Mg2+
Concentradon in working mix (uM)
Specific targets?
518F 64 10 Universal 926R1 52 8 Rikenella 926R3 57 1 926R4 55 1 Epsilons
Bacterial v4v5
Thermocycling: 94˚C, 3:00 25 cycles of: [94˚C, 0:30; 55-‐60˚C, 0:45; 72˚C, 1:00] 72˚C, 2:00 4˚C, hold
454 v6v4 and MiSeq v4v5 datasets
Sewage 19_SS 1 step 2 step Bv6v4
20_SS Bv6v4 21_SS Bv6v4
Leech I_GS1_R2 Bv6v4 REA_HDF Bv6v4
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OPTIMIZING PCR CONDITIONS
or, how Sharon spent her spring
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CondiKons to try
• Primer concentraKon – 0.2-‐0.4 uM
• Magnesium concentraKon – 1.5mM – 3.67mM
• Annealing temperature – Tm of primers suggest anywhere from 52˚C-‐60˚C
• Keep primer composiKon and cycle number constant
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Leech
Ladd
er
sewage
Ladd
er
REA_HDF
55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C
0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3
A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B
Effect on amplificaNon of
Condidon Leech REA_HDF
Increase temperature Decrease Decrease
Increase primer Increase Increase
Increase magnesium Increase Increase
Annealing temperature, Primers, A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 All NTC at 55˚C
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19_SS
Ladd
er
20_SS
Ladd
er
21_SS
55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C
0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3
A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B
Effect on amplificaNon of
Condidon 19_SS 20_SS 21_SS
Increase temperature Decrease Decrease Decrease
Increase primer Increase Increase Increase
Increase magnesium Increase Increase Increase
Annealing temperature, Primers, A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 All NTC at 55˚C
(+)/(-‐)
55˚C 57˚C
0.2 0.3 0.2 0.3
A B A B A B A B
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Arco_1
Ladd
er
Arco_3
Ladd
er
209_5M
55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C
0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3
A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B
Effect on amplificaNon of
Condidon Arco_1 Arco_3 209_5M
Increase temperature Decrease Decrease N/A
Increase primer No Effect Minor increase N/A
Increase magnesium Increase Increase N/A
Annealing temperature, Primers, A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 All NTC at 55˚C
(+)/(-‐)
55˚C 57˚C
0.2 0.3 0.2 0.3
A B A B A B A B
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Archaeal v4v5 • Try annealing temperature, primer concentraKon, magnesium
concentraKon • Hold constant 1X HiFi Buffer, 0.1 U Taq/uL, template • Use Av6 and Bv6 as posiKve control for template
Primer Tm at 1.5-‐3.6mM Mg2+
Concentradon in working mix (uM)
arc517F1 63 10 arc517F2 59 10 arc517F3 58 10 arc517F4 56 10 arc517F5 56 10 arc958R 57 10
Thermocycling: 94˚C, 3:00 30 cycles of: [94˚C, 0:30; 55-‐60˚C, 0:45; 72˚C, 1:00] 72˚C, 2:00 4˚C, hold
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Av4v5
Ladd
er2
Av6 Bv6*
55˚C 60˚C
NTC
55˚C 60˚C
NTC
60˚C
NTC
0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3
A B A B A B A B A B A B A B A B A B A B
Annealing temperature, Primers, MgSO4 A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 1X HiFi Buffer, 0.1 U Taq/uL. All NTC at 0.2 uM each primer, 1.5 mM MgCl2, 55˚C
Effect on amplificaNon of
Condidon Av4v5 Av6 Bv6*
Increase temperature Decrease No change N/A
Increase primer Increase Increase Increase
Increase magnesium Increase Increase Increase
Thermocycling: 94˚C, 3:00 30 cycles of: [94˚C, 0:30; 55-‐60˚C, 0:45; 72˚C, 1:00] 72˚C, 2:00 4˚C, hold
*In comparison, the usual cocktail has 1X HiFi Buffer 2 mM MgSO4 0.2 mM each dNTP 0.4 uM each primer 0.02 U/uL Taq Plenty of DNA In 33 uL total
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Arcobacter
Epsilons Rikenella
Primer Tm at 1.5-‐3.6mM Mg2+ Specific targets?
518F 64 Universal 926R1 52 Rikenella 926R3 57 926R4 55 Epsilons
Component 454 Bv4v5 Bv6 Av6 Av4v5
X HiFi 1 1 1 1 1
mM MgSO4 3.67 2 2 1.5 1.5
mM dNTPs 0.2 0.2 0.2 0.2 0.2
U/uL Taq 0.1 0.1 0.02 0.1 0.1
uM each primer (set) 0.3 0.4 0.2 0.3 0.3 annealing temp 60 60 60 60 55
cycles 30 25 25 25 30
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If you see your products amplify with less Taq... tell us your secrets.
2 mM Mg2+, 0.02 U/ul Taq, 0.2 uM primers
3.67 mM Mg2+, 0.1 U/ul Taq, 0.37 uM primers
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Current protocols bac v6 arc v6 bac v4v5 arc v4v5
HiFi buffer 1X 1X 1X 1X MgSO4 2 mM 2 mM 3 mM 3 mM dNTPs 0.2 mM 0.2 mM 0.2 mM 0.2 mM
combined primers 0.2 uM 0.3 uM 0.3 uM 0.3 uM PlaKnum HiFi 4 units 10 units 10 units 10 units Template 5-‐20 ng 5-‐20 ng 5-‐20 ng 5-‐20 ng Volume 100 ul 100 ul 100 ul 100 ul
PCR 3min 94o 94o 94o 94o 30 sec 94o 94o 94o 94o 45 sec 60o 60o 57o 57o 1 min 72o 72o 72o 72o 2 min 72o 72o 72o 72o
Cycles, step 1 25 30 30 30
Pooling, clean up Qiagen MinElute
plate Qiagen MinElute
plate Ampure, 1:1 Ampure, 1:1
Step 2 4 ul step 1 eluate 0 0 0 Cycles, step 2 5 0 0 0 QuanKtaKon Picogreen Picogreen Picogreen Picogreen Size selecKon Pippin Prep Pippin Prep Ampure Ampure
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Typical amplificaKon results
4900
1900 2900
100
1100
300
500 700
Archaeal v4v5 pooled, on Caliper GX Bacterial v6 pooled, on Caliper GX
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Size selecKon
v6 amplicons (Pippin)
v4v5 amplicons (Ampure)
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Illumina amplicon QC
• Filter out reads based on: – ChasKty filter – Ns
• Then ajempt to merge forward and reverse reads • Keep only those that match perfectly over the region of overlap (v6) or have fewer than 3 mismatches (v4v5)
• Create consensus using basecall with higher Qscore • Trim away adapters, primers • Product goes into usual GAST pipeline, but as unique sequences plus frequency info, not all reads
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2012-‐13 amplicon sequencing goals
• Complete 454 projects in progress √ • ReKre GS-‐FLX √ • ConKnue to use HS1000 for metagenomics, short reads, V6 √ • Use 96+ mulKplexed V6 amplicons on HiSeq (paired end
perfect overlaps) √ • Use mulKplexed V4 amplicons (no overlap) X • Run V4-‐V5 amplicons on MiSeq (parKal overlap for QC and
merging) √ • ReKre PGM √
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Final comments • Amplicon sequencing is moving target
– Not as well supported by next-‐gen companies as shotgun sequencing – Databases growing – Sample sources expanding (animal microbiomes, extreme environments, low
biomass targets, marine/freshwater/sediments/rock surfaces) – Read lengths increasing; paired end reads possible – Processing s/w –many approaches to filtering, trimming
• So— – Look at your raw data – Assess expected coverage by primer sets – OpKmize amplicon protocols and processing – Evaluate accuracy, not just quality (run controls) – Don’t hesitate to discard LQ reads – Expect technological advances