Preliminary report describing a targeted sequencing study in 1500 MS cases and 1500 controls. I presented this at the ASHG 2011 session on large scale resequencing.

1. Next generation sequencing in
multiple sclerosis
Chris Cotsapas
Neurology/Genetics @ Yale
cotsapaslab.info

2. Acknowledgements
• John Murdoch
• Cristin Aubin
• Stephan Sanders
• Matthew State
• Phil De Jager
• David Hafler
• Yale Keck YCGA; Mane Shrikant

3. Multiple sclerosis

4. MS NGS, oh yes
• Discover variation in/around 35 MS candidate
genes in 1500 cases and 1500 controls
• Validate hits in 10,000 cases and 10,000
controls
• Test hypotheses of overall and pathway
burden, common/rare combinations

5. Targets
IL23R CD58 RGS1 KIF21B CXCR4 TMEM39A
IL12A IL7R PTGER4 IL12B TNFAIP3 IL7
JAK2 MLANA IL2RA ZMIZ1 NKX2.3 CD6
SIAE TNFRSF1A MGST1 IL23A SH2B3 PTPN11
MPHOSPH9 CDK2AP1 CLEC16A SOCS1 IRF8 STAT3
TYK2 CD40 OLIG3 TBRG1 SPA17
RNF40
STX1B
KIF21B
STX4
CACNA1S
P4HA2
PHKG2 SETD1A
SRCAP
ITGAL RAD50
IRF1 ZNF768
STAT5B EBF1
STAT5A
IL12B
IL5 STAT3
CSF2
CTF1
IL3 JAK2
INSL6
IL23R
C1orf106 IL12RB2
PTRF
Rossin et al, Cotsapas et al PLoS Genet 2011 DCTPP1

6. Experimental design
John Murdoch, Cristin Aubin

7. Conservative MAF representation with
different pool sizes
14.00%
12.00%
10.00%
8.00% 2.59%
Min. Allele %
6.00% Mean Allele %
4.00%
2.00%
0.00%
0 2 4 6 8 10 12 14 16 18

8. Raindance technology

9. GERALD
Align to
reference
Convert to
SAMtools
Trim to Target
Coverage
Sort and Pileup
Error Rate Filter SNPs
Results Annotate
Stephan Sanders

10. State of play: 656 cases, 640 controls

11. Summary statistics (per pool, with sd)
• Barcoded reads passing filter per pool: 21 mil (6.5 mil)
• Mean coverage per pool: 3066x (649x)
• % bases > 80x coverage 97.7 (1.2)
• % bases on-genome: 87.8 (1.4)
• % bases on-target 66.7 (6.0)
– drop-off due to residual genomic DNA
• Mean error rate: 0.51% (0.1%)
• Ti/Tv = 3.97

12. Summary statistics by status
Case [mean(sd)] Control [mean(sd)]
Sample N 656 640
Total passed reads/pool 21.1 million (6.9 mil) 20.9 million (6.2 mil)
Mean coverage/pool 3063x (680x) 3069x (619x)
Lane representation 49.9% (7.0%) 50.2% (7.0%)
% bases 80x 97.6% (1.0%) 97.9% (1.3%)
% bases on-genome 87.9% (1.3%) 87.6% (1.5%)
% bases on-target 66.9% (5.1%) 66.4% (6.9%)
Error rate 0.51% (0.10%) 0.51% (0.09%)
John Murdoch

13. C-alpha distributional testing
• Test categories
– Missense/splice variants < 5% MAF
– Silent variants < 5% MAF
– All singletons
Gene M/SS M/SS Silent Silent Singleton Singleton
counts Calpha p counts Calpha p counts Calpha p
JAK2 2/7 0.04 408/401 0.91 34/45 0.18
IL2RA 4/0 0.05 94/75 0.19 17/16 0.86
NKX2-3 1/1 0.50 691/660 0.69 3/8 0.06
IRF8 1/3 0.12 211/222 0.42 35/13 0.0016
Stephan Sanders, Ben Neale

14. Future work
• Collect remaining data
• PCR-based validation
– Per-sample genotypes
• Replicate in 10K/10K cohort (Q2/2012)
• GWAS/Immunochip integration
– Condition on common variants
• Pathway burden analysis

15. Last impressions