1. Considerations for Analyzing Targeted NGS Data BRCA Tim Hague,CTO

2. Introduction  BRCA 1 and 2 are best known as 'cancer susceptibility' genes  Actually the proteins repair damage in DNA  Large number of known deleterious mutations  Disproportionate number of indels

3. History  Mary-Claire King discovered BRCA1 and BRCA2, published the function  Myriad Genetics won the patent

4. Distribution of known BRCA1 deletions >3 bp Indel size (nt)

5. Dominuque Stoppa Lyonnet at Curie Institute „Large scale deletions could account for as many as one-third of all BRCA1 mutations in some populations”

6. BRCA are tumor suppressor genes. 82% lifetime chance of developing breast/ovarian cancer. Science 2004, 306:2187-2191 >1,500 deleterious BRCA mutations 17 kbp coding region with mutation rate of 1/2000 NGS-based BRCA screening Leeds UK, Newgene UK, Ghent Belgium DIY genetic test published by Salzberg

7. 82% chance of cancer >90% chance of being false positive/ negative

9. What kind of NGS data?  False negatives must be avoided  Precision of both sequencing data and the data analysis is key  Looking for indels – indel detection abilities are a key criterion  Repeats are also an issue in BRCA region

10. BRCA Repeats

11. Homopolymer Errors Homopolymer errors look like small indels and can cause noise Problem for:Roche 454 Ion Torrent

12. Long Reads Read length is a limiting factor for insertion detection. When searching for indels, long reads can help. Long reads can also help with repeats. Roche 454 have the longest reads.

13. Real examples with Roche 454 data

15. Paired Reads  Paired reads can also help to increase effective 'read length'  Illumina MiSeq now has 2x250bp protocol

17.  Compare 9 open source and commercial NGS analysis softwares  In silico test with mutated reference BRCA gene  2211 known BRCA variants 1341 SNOs, 320 insertions and 551 deletions  Full GATK pipeline used for variant call, including quality recalibration and indel realignment

21. BWA Overall Sensitivity: 99.2% Paired End 94.5% Single End SNPs found: 99.5% PE 99.5% SE Deletions found: 98.5% PE 85.5% SE Insertions found: 99.4% PE 89.4% SE

22. BWA False Negatives: 17 Paired End 121 Single End False Positives: 23 PE 168 SE The longest (60bp+) deletions were not found, either with PE or SE data

23. Indel sizes - BWA Single End

24. Indel sizes - BWA Paired End

25. Other Tools  Most other alignment tools showed a similar trend – much better results overall with Paired data  Only two of the tools tested found the longest deletions, even with Paired data

26. Paired Reads - Conclusions  Much better for reliable variant detection than equivalent length single reads  Provided much better coverage in the BRCA region (spanning small repeats) If available, paired reads should be preferred

27. Indel Detection - Conclusions  Not all tools are good at finding indels.  Burrows Wheeler based aligners can't find indels beyond a few base pairs in single reads, but can make better use of paired data – if indel realignment is also used.  They still can't detect the longest indels (there is just a gap in coverage). If indel detection is required, an indel sensitive tool should be used

28. Overall - Conclusions  None of the alignment tools found all the variants  It will almost certainly require the same data to be analyzed with more than one tool, to get sufficiently accurate results