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Lab Week Party Kits

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Lab Week Party Kits

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Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:https://www.surveymonkey.com/r/LabWeekImmucor2020

• Registration deadline is May 8, 2020

• Certificates will be sent via email only to those who have registered May 22, 2020

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• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.

• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.

NGS and New Alleles, now what?

Michael D. Gautreaux, Ph.D., D.ABHIKelly J. Ingram, BS, MS CHT

David F. Kiger, BS, CHS

Happy Lab Week!

#ASHILabWeek2020

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Disclaimers

• I will strive not to give the appearance of favoring one vendor over another.

• You will not be able to leave this webinar and be able to perform NGS typing in your lab.

– That’s true for any technology used in our field.

Learning Objectives

1. Realize that the process for naming new HLA alleles involves multiple steps.

2. Understanding the required elements for successfully submitting novel HLA alleles for official naming with IPD-IMGT.

3. Adapt ideas from this lecture into your everyday practice.

HLA DNA Bible

https://studyres.com/doc/6443673/dna-methods-for-hla-typing-a-workbook-for---ashi-u

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What exactly is NGS?

• The standard definition– Next-Generation Sequencing is a high-throughput

DNA sequencing methodology that makes use of parallelization to process up to half a million sequences concurrently.

• Popular definition– A new era in DNA testing. Whole genome

sequencing is an area of increasing popularity especially in “personalized medicine”.

HLA and Personalized Medicine

• A person’s HLA represents self to the immune system. What’s more personalized than self?

– Amy Hahn, Ph.D., D.ABHI

ASHI Meeting 2015

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SBT & NGS Amplicons

GenDx HLA Typing by Next-Generation Sequencing Education Booklet

Reagent costs per patient

Description

Reagent and Supply 

Cost Per Patient

Sanger

Overhead $18.12

Tech costs $27.41

Consumables $90.08

Reagent Kits $348.62

Machines* $45.75

* = 10 year 

depreciation

Low Res Costs 401.59

Sanger Costs 529.98

Total SBT Costs 931.57

NGS

Overhead 18.12

Tech costs 36.55

Consumables 75.79

Reagent Kits 100.00

Machine** 8.01

** = 10 year 

depreciation

Total NGS Costs $238.47

Get Docs to help

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NGS Terms to Know

• Resequencing– term for sequencing an region that has already been

sequenced. Reads need only be a few hundred base pairs long. Many Next-Generation Sequencing platforms provide short reads that can be aligned to a reference genome.

• De novo sequencing– term used to sequence a genome from scratch. It is

much more costly, time-intensive, and limited to select techniques. The length of a read must be at least 1,000 bps long. The first human genome sequenced relied on these methods, which is one of the reasons it was so costly and time-intensive.

NGS terms

• Read– A raw sequence that comes from a sequencing

machine. Usually 300-800 bp long.

• Tag– Several reads coming from the same sequences can

be merged to one tag.

• Sequencing Depth– Total number of sequences, reads, or base pairs

generated represented in a single sequencing experiment.

• Coverage– Total number of bases generated / size of genome

sequenced.

MIA FORA Coverage

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General Workflow for NGS

1. Target Generation– HLA locus of interest is amplified

2. Library Preparation– Prepared according to platform of choice– Fragmentation, adapter ligation and barcoding

3. Clonal Amplification – Generates sufficient material for detection– Not required for PacBio & Oxford Nanopore (SMRT)– Illumina performs bridge amplification

4. Sequencing– Sequencing by ligation (Thermo-Fisher), sequencing

by synthesis (Roche 454, Ion Torrent, Illumina, Qiagen)

5. Data analysis– Varies by vendor and machine

MIA FORA MFlex Workflow

Unusual Results

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Novel Allele?

Submission Process

1. Prepare Submission Files 2. Submit Files to GenBank3. Receive Accession Number4. Submit to IMGT Database5. Receive Novel Allele NamePrepare

submissionGet new

allele name

IPD-IMGT/HLA Database

• IPD-IMGT/HLA Database Submission Tool– https://www.ebi.ac.uk/ipd/imgt/hla/subs/submit.html– allows direct submission of sequences to the WHO HLA Nomenclature

Committee for Factors of the HLA System– there are required conditions for the acceptance of new sequences for

official names

• An accession number in a sequence databank must have been obtained. Sequences may be submitted to these databases online at the following addresses:

– EMBL: /embl/Submission/index.html (European)– GenBank: http://www.ncbi.nlm.nih.gov/Genbank/submit.html (US)– DDBJ: http://www.ddbj.nig.ac.jp/submission-e.html (Japan)

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Preparing Databank Submission

• Vendor-dependent– All vendors offer support to confirm potential new alleles

• FASTA/FASTQ– Vendors’ software generate the sequence

• Identify coding and non-coding regions & the position of the variant codon(s)

– 5 column feature table» gives general features of the gene

– Blast» Gives position of variants

Files

• FSA File (FASTA)-Nucleotide Sequence

• TBL File-Table of Exon/Intron Information

Submit Files to GenBank

• https://www.ncbi.nlm.nih.gov/WebSub/index.cgi

• Follow Prompts to add: Authors Sequencing Technology (i.e. Illumina, IonTorrent, etc.) Assembly Program (NGSengine) Release Date Molecular Type (genomic DNA)

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GenBank

BankIt

BankIt Welcome

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BankIt Information

BankIt Authors

BankIt Technology

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BankIt Sequence Data

BankIt Modifiers

BankIt Features

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BankIt Features 2

BankIt Review

The Accession Number!

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Finished GenBank Submission

• Next step is to start IMGT submission

• Need same data as GenBank submission

Welcome to IPD-IMGT/HLA

• https://www.ebi.ac.uk/ipd/imgt/hla/

Submission Tool

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Submission Checklist

x

x

x

x

x

x

x

Submitter Information

Stage 2

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Stage 3

Stage 4

Stage 5a

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Stage 5b

Derived from TBL

FSA file

Stage 6

Derived from TBL

Stage 6 (cont.)

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Stage 7

Stage 8

Stage 8 (cont.)

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Stage 9

Done!

Confirmation E-mail

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The Letter

WF HLA Publications

10 alleles

28 alleles

Loci of novel alleles

HLA Locus

# of alleles thru 12/19

A 4B 2C 4

DRB1 1DRB3 2DRB4 0DRB5 0DQA1 11DQB1 0DPA1 9DPB1 5

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Number of alleles discovered/month

1 – 1.5% rate!

Conclusions

• (NGS) is revolutionizing immunogenetics – significant reduction in errors compared to Sanger-based SBT – increase in the discovery of novel HLA alleles.

• As more labs begin to adopt this technology, – the discoveries of new alleles will continue– rate of such discovery is unknown

» reporting new alleles is difficult and not mandatory

• Currently ~26,000 HLA alleles– How many alleles would be problematic?

» 50K, 100K, 1 million?

• Process of submitting a novel allele for naming – Too time-consuming and labor-intensive– needs to be simplified or automated

Thanks!

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Wake Forest HLA/Immunogenetics Laboratoryhttps://www.wakehealth.edu/Specialty/p/Pathology/HLA-Immunogenetics-Laboratory

We’re on

• Director –– Michael D. Gautreaux, Ph.D., CHT, D. ABHI

• Supervisor –– David F. Kiger, BS, CHT, CHS

• Technologists –– Elaine F. O’Shields, BA, CHT, CHS– Jennie W. Stewart, BS, MT(ASCP), CHT, CHS– Sharlie B. Brown, BS, CHT, CHS– Kimberly N. Weavil, BS, MT(ASCP), CHT, CHS– Kelly J. Ingram, BS, MS, CHT– Eric M. Netherton, BS, CHT

• Laboratory Assistants –– Marcia Bassette & Aneesah Harris

• Office Coordinator –– Patti Shew

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Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:https://www.surveymonkey.com/r/LabWeekImmucor2020

• Registration deadline is May 8, 2020

• Certificates will be sent via email only to those who have registered May 22, 2020

Seeing Beyond Limits

2020 Webinar Schedule

Link to register:  https://immucor.webinato.com/register

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Seeing Beyond Limits

2020 “Mini” WebinarsShort,10-minute educational videos with an industry leader.

Link to register:  https://immucor.webinato.com/register

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Thank you!