This document describes a bioinformatics workflow to annotate somatic mutations with four confidence levels. It involved analyzing variants called from 63 pairs of tumor-normal BAM files from 21 replicates across 6 institutions. Variants were classified using SomaticSeq and classifications were consistent across sequencing replicates and aligners. The confidence levels and variant allele frequencies were broken down, and VAF peaks corresponded to copy number states while shifts corresponded to copy number changes. A subset of calls were validated using AmpliSeq and Ion Torrent sequencing, and limitations included unmappable regions, very low VAF variants, and complex variants. The workflow resulted in a comprehensive somatic mutation call set across the genome down to ~5% VAF.

2. Bioinformatics workflow to annotate four confidence levels
21 replicates
6 institutions 63 pairs of BAM files Variants called and scored in each tumor-normal pair
cross-aligner and
cross-institution
reproducibility

3. SNV indels
AmpliSeq validated
SomaticSeq classifications consistent across sequencing replicates and aligners

4. Breakdown of confidence levels and VAFs

5. VAF peaks correspond to Copy Number states
For Research Use Only. Not for use in diagnostic procedures. 5
1/1 2/2
1/2
3/3
2/3
1/3
2/4
1/4
4/5
2/5
1/5
2/6
3/6
4/6
5/6
1/6

6. VAF shifts correspond to Copy Number changes
6

7. SNV INDEL
AmpliSeq Validation (Depth ≈ 2000X)
For Research Use Only. Not for use in diagnostic procedures. 7
Validated Invalidated
X Uninterpretable (low depth or unclear signal)
Validation Rate =
+
Total Number | Interpretable | Validation Rate

8. 3/247 HighConf Calls “Negatively Validated”
1. Weak but clear variant signal in Normal ( x2 )
2. Strong variant signal everywhere else ( x1 )
7/105 Unclassified Calls “Positively Validated”
1. Low mapping quality scores ( x3 )
2. Part of di-nucleotide change ( x2 )
3. Adjacent to large deletions ( x2 )
Discrepancies between Super Set and AmpliSeq
For Research Use Only. Not for use in diagnostic procedures.8
Normal VAF
(Gold Set)
Normal VAF
(AmpliSeq)
i) 0.7% 12.4%
ii) 5.0% 3.2%
AmpliSeq
0
0.1
0.2
0.3
0.4
0.5
0.6
VAF
BWA
Bowtie2
NovoAlign

9. SNV INDEL
Ion Torrent Validation (Depth = 34X )
For Research Use Only. Not for use in diagnostic procedures. 9
Validated Invalidated
X Uninterpretable (low depth or unclear signal)
Validation Rate =
+
Total Number | Interpretable | Validation Rate

10. Summary
• Vast majority of calls either have broad support everywhere, or not much at all
• Limitations
• Unmappable (by short-reads) regions
• Very low VAF
• Complex variants, i.e., multi-nucleotide variants
• Adjacent indels
• Comprehensive somatic mutation call set
• Across (most of) the entire genome
• Sensitive and accurate down to ~ 5% VAF
For Research Use Only. Not for use in diagnostic procedures.10
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The Somatic Mutation Working Group
Government Industry Academic
EPA Abbvie Cornell University
FDA/CBER AstraZenca EATRIS
FDA/CDER ATCC Fudan U.
FDA/CDRH DNANexus Garvan Institute
FDA/OC Du Pont HudsonAlpha
FDA/NCTR Genentech Indiana U.
NIH/NCBI Illumina LLU
NIH/NCI MedImmune Mayo Clinical
NIH/NHLBI Novartis MD Anderson
NIH/NIDCD PacBio U. Toledo
NIH/NIEHS Roche U.of Cologne
USACE Seven Bridges Yale
~200 members
60+ institutes
Participants: Wenming Xiao FDA
Huixiao Hong FDA
Yongmei Zhao NCI
Daoud Meerzaman NCI
Charles Wang Loma Linda U.
Li Tai Fang Roche
Mehdi Pirooznia NHLBI
Chunlin Xiao NCBI
Leming Shi Fudan U.
Leaders: