ADNI Sequencing Working Group. Robert C. Green, MD, MPH Andrew J. Saykin, PsyD Arthur Toga, PhD

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1 ADNI Sequencing Working Group Robert C. Green, MD, MPH Andrew J. Saykin, PsyD Arthur Toga, PhD

2 Why sequencing?

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4 V V V V V V V V V V V V V

5 A fortuitous relationship

6 TIME s Best Invention of 2008

7 The initial proposal Spring, 2012 We requested $10 million from Brin-Wojcicki Foundation to sequence, distribute and analyze approx 800 samples

8 $2 million June, 2012

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10 ADNI Cases Sequenced N = AD MCI Controls Unstable

11 What is involved in sequencing?

12 GENERAL NGS WORKFLOW FRAGMENT Sonication (e.g. Covaris) Enzymatic digest (e.g. Haloplex,Agilent) Transposable elements/digest (e.g. Nextera, Illumina) Other Sample Prep (Library) ADD SEQUENCING ADAPTERS SELECT TARGETS In solution Solid arrays capture (Agilent) MIPs Other (Nimblegen) Target Selection (Enrichment) Illumina HiSeq MiSeq Life SOLiD Ion Torrent SEQUENCE Roche rd Gen Primary Analysis BWA NOVO ALIGN ALIGN + CALL VARIANTS STAMPY NEXT GENE MANY OTHERS GATK XXX YYY Secondary Analysis INTERPRET SEQUENCE VARIANTS Tertiary Analysis

13 SHORTS READ ASSEMBLY INTO SEQUENCE THROUGH ALIGNMENT TO REFERENCE Reference Sequence NGS reads Aligner (BWA, Novoalign, etc) NGS reads are short (though technology is improving) alignment is difficult and error prone

14 SAMPLE OUTPUT (BWA) Sanger Heterozygous C>T Illumina NEXT GEN IS QUANTITATIVE, COUNTS # TIMES A VARIANT IS SEEN Coverage = 20x Allelic ratio = 0.55 (C = 9; T = 11)

15 COVERAGE HOW MUCH DO YOU NEED? Example (germline disease) - 46 genes (~250 kb) 10 samples/lane (HiSeq2000) Coverage threshold: 20x Any base <20x Sanger >97% of bases >20x (most have excessive coverage) Don t really need 400x cov but found that sequencing at that depth boosts problem regions 3 % with zero cov Can affect many exons

16 Charge to the ADNI Sequencing Working Group Prepare these data to share with qualified investigators!

17 Analyst Data

18 Charge to the ADNI Sequencing Working Group Prepare these data to share with qualified investigators! Avoid ethical and PR disasters!

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21 ADNI WGS Timeline

22 Tasks of the Sequencing Workgroup? Review consent language Organization, QC and Processing Prepare the data for sharing Sharing with NIAGADS Sharing with qualified investigators Share the data

23 Tasks of the Sequencing Workgroup? Review consent language Organization, QC and Processing Prepare the data for sharing Sharing with NIAGADS Sharing with qualified investigators Share the data

24 Consent Analysis

25 Consent Analysis (N = 502 Forms) Database Overview of findings by site, protocol and consent dates. Corresponding archive of language used in consent forms (CFs). What samples or data are specified as sharable? With whom may samples or data be shared? For what kinds of research will samples or data be used in the future? Additional constraints? Findings: CFs either allowed all sharing or provided checklists for participants to select what may be stored and shared. Findings: All CFs provided for sharing with ADNI and the general scientific community. Findings: Most CFs suggested types of research and had a general statement about future sharing. Some CFs used restrictive language (i.e., will only be used ) Findings: One site allowed participants to opt out in advance, for when they lose capacity. Following up with sites that used checklists to determine participant selections. No action needed. Followed up on findings of restrictive language. Determined that storage and sharing are not affected. 1 site (5 participants) affected, and follow up is in progress.

26 Tasks of the Sequencing Workgroup? Review consent language Organization, QC and Processing Prepare the data for sharing Sharing with NIAGADS Sharing with qualified investigators Share the data

27 What do we get from Illumina?

28 What do we get from Illumina? Variant (vcf) files (< 1 GB) Raw (bam) files (100 GB) Supplemental files (2-5 GB) Omni 2.5 genome scan Realigned segments Index files Annotations

29 What do we get from Illumina?

30 Receiving and Organizing ADNI WGS Data Where to put it?

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32 ADNI Cases Sequenced Institution Samples Received to Date LONI 845 (100%) Broad/HMS 754 (89%) Indiana 771 (91%) NOTE: N > 818 due to some file errors that were re-done.

33 Tasks of the Sequencing Workgroup? Review consent language Organization, QC and Processing Prepare the data for sharing with NIAGADS with qualified investigators Share the data

34 Organization, QC and Processing First Tier Check initial integrity of data File integrity check: confirming gender, APOE with existing ADNI data Comparison of vcf files and new Omni scans with existing genotype data Create tools for annotating, partitioning, selecting, querying and distributing data. Explore strategies to extract detailed sequenced regions for targeted analysis Develop basic data distribution mechanisms

35 Organization, QC and Processing Second Tier Assess Casava alignment against best practices and consider re-alignment Assess proportion of known variants in the set of allelic variant calls, test agreement of statistical properties of variant calls with known properties of naturally occurring human mutations. Compare statistical properties of the variant call set to known biases in human polymorphism data. Analyze distribution of allele frequencies and the proportion of variants seen in both chromosomal copies as opposed to variants seen in only 1 copy.

36 Tasks of the Sequencing Workgroup? Review consent language Organization, QC and Processing Prepare the data for sharing Sharing with NIAGADS Sharing with qualified investigators Share the data

37 Plan for Sharing the Data Internet-based sharing of vcf files Physical access to data in 3 geographic locations: LA, Indianapolis, Boston Purchase and shipping of large hard drives: the lending library approach Storing genome data in the cloud (expensive) Remote access to stored data (experimental) New forms of data compression (experimental)

38 Imagine if ADNI funding only supported data production

39 All Sequencing Working Group Activities are Currently Unfunded Created a budget of $1.1 million submitted to BWF in July, 2012 received encouraging feedback. Three days ago, BWF offered $300K as a challenge if the remainder can be raised from other sources. Requesting consideration of matching $300K from the 26 members of PPSB. Will seek remaining $400,000 from other sources.

40 Thank you! Questions for the Sequencing Workgroup? genomes2people.org