# Presentation: 902505

## Updating Strategiesfor Mutation Detection in TCGA

**Human Genome Sequencing Center**
**Baylor College of Medicine**

## Natural Life of a Technology:

- Proof of concept (***promising***)
- Demonstration of real utility (***really works***)
- Application
- Optimization/production (***good***)
- Optimization/production (***better***)
- Optimization/production (***even better***)
- Optimization/production (***near perfect***)
- Retire/replace (***gone***)

## Transitions:

**1989 – PCR/Sanger fluorescent sequencing**
**2006- 2008 ~6M Medseq Reads**
***2005 – first NexGen data***
***2007 – Watson Genome sequence***
***2007 – First capture data***
***2008 – Capture optimization phase 1 and 2***
***2008 – First whole tumor genome sequences***
**2009 – plan to begin year with ‘downsized’****            PCR/MedSeq data **

## HGSC MedSeq Production

**Sunset beginning **
**   ****Jan 2009?**

## ‘Project Jim’: A Personal Genome

- What can we learn from a single genome?
- 454 Data
- 234 runs on FLX (~ 2 months, $1-2M)
- ~ 106 million reads -8 x coverage

**‘****Project Jim’: A Personal ****Genome**

## Chromosome 1 base coverage: p arm

***centromere***

**Chromosome 1 (Mb)**

**Coverage in 5kb windows across p arm of Chromosome 1**

**Notes:**
Coverage across chromosome 1 is measured in consecutive 5K windows. Red dots are regions of *** or more windows with coverage less than 3.

## Copy number variation

- ...CACAGT-GCAGAACATTTGGAGCTGCTCCATGC**AG**catccctgggatgcaaggctggttcaacaca...
- ...CACAGTAGCAGAACATTTGGAGCTGCTCCATGC**AG**catccctggg
- ...CACAGT-GCAGAACATTTGGAGCTGCTCCATGC**AG**catccctggg
- ...CACAGT-GCAGAACATTTGGAGCTGCTCCATGC**AG**catccctgggatgcaaggctggttcaacaca...
-                                  TGC**AG**catccctgggatgcaaggctggttcaacaca...

**Notes:**
Breakpoints at 150,822,165  150,854,364

## Deletions in 454 reads

- 157,041 deletions;
- 65,677 insertions: 2-208 bp

## Evaluation of errors in 454

- ...AG**C****T**TTTT...    read
- ...AG**T****C**TTTT...    reference
- Swapped (MNP) base

- 0.00    0.005    0.010     0.015

- Distance to 3’ end

- 0       100      200      300     400

- 0        10        20         30        40

- 0.0  0.05  0.10  0.15  0.20  0.25

- Quality

- Proportion of SNPs

- Proportion of SNPs

- Homopolymer Run

- Homopolymer run

**Notes:**
Figure 1. Logistic regression to distinguish 454 systematic errors with true SNPs.
A) Homopolymer runs vs substitution errors.
B) The quality score distribution of “True” and “False” SNPs.
C) The distribution of distances to 3’ end of 454-FLX reads. 
D) The distribution of l_p from logistic regression model.

## Single nucleotide variation in JDW 454 reads

- Watson, 7.4X “Pyro”;   Venter, 7.5X “Sanger”
- Filters:
- Raw, all base substitution from cross_match alignments
- 1, Sv >28
- 2, filter 1 plus ratio of variant to total coverage 0.2
- 3, filter 2 plus eliminate SNPs close to homopolymer runs >5 bp.
- 4, (Venter) Phred Q >15, ratio of variant to total coverage 0.2

**Notes:**
This table contains a reassessment of the Venter SNP numbers, which aren’t clearly stated in any single table. The data in Table 3 from which we have drawn some numbers actually contain total variation of all kinds.  So I have recalculated the Venter numbers from data in table 4 (Internal HuRef-NCBI Map column), table 6 and table 3 as follows: Table 4 gives the tallies for the “high confidence” variation.  Sum the tallies of SNPs and MNPs and convert MNP tallies to individual SNP counts using average MNP lengths from Table 6 (since our tallies are consider each SNP independently). Use 82% as fraction from Table 3 to obtain known SNPs and subtract for novel SNPs.

## Validation of 454 SNPs by SNP array

- False negative rate for heterozygotes in JDW is similar to Sanger sequence of Venter

**Notes:**
Need more explanation of the comment on False positives. 

## Recovery of heterozygotes

- Coverage follows Poisson distribution
- Het discovery follows binomial
- Variant requires two observations

- 7.4X

**Notes:**
Supplementary Figure 4. Detection of heterozygotes as a function of WGS coverage. The theoretical yield of heterozygotes as a function of coverage was calculated as described in Methods “Filtering Criteria for SNPs”. 

## 454 sequencing in validation of SNPs and indels

- 100-200 PCR amplicons, pooled and sequenced on 454
- Differs from WGS in depth of coverage (200-2000x)
- Used in validation of tumor mutations as well as germline polymorphism

## Average Pr(e|c)

## Post Watson Developments: 

**Post Watson Developments: **

**Installed 10 FLXs 454 machines in **
**Sept 07 Converted 5 to XLR capable**

**Installed 6 SOLiD machines June ’08****Initially for 1000 Genomes project **

## 		454 FLX – In Production for ~1 year
18 runs/day by July 30
Total of 1,365 runs for 140Gb of Sequence
i.e average > 100 Mb/run

		454 XLR/Titanium - In Evaluation 
68 runs for 23Gb of Sequence
Best run - 562 MB, 1.5M pass filter reads

- 18 runs/day by July 30
- Total of 1,365 runs for 140Gb of Sequence
- i.e average > 100 Mb/run
**	****	****454 XLR/Titanium - In Evaluation**** **
- 68 runs for 23Gb of Sequence
- Best run - 562 MB, 1.5M pass filter reads

## SOLiD 3kb Paired end Read Production Projections

|  | April | May | June | July | Aug |
| --- | --- | --- | --- | --- | --- |
| Runs | 2  /  0 | 8  /  6 | 12  /  8 | 12  /  8 | 12  /  8 |
| GB/Run | 4  /  0 | 4  /  10 |  5  /  12 |  5  /  12 |  5  /  12 |
| Slides | 4  /  0 | 16  / 11 | 24  /  20 | 24  /  20 | 24  /  20 |
| GB | 8GB/0GB | 32GB/ 56GB | 60GB / 96GB | 60GB / 96GB | 60GB / 96GB |

- Original projected capacity - 220GB
- Current projected capacity - 344GB

## GBM Whole Genome Sequencing Plans:

**GBM Whole Genome Sequencing Plans:**

**Tumor:**
**454 – 36 Gb read coverage**
**SOLiD ~ 200 x 34kb clone coverage (9 Gb seq)**
**Normal: **
**6 x 454 coverage **
**Data generation Nov ‘08 – Feb ‘09**

## Nimblegen Capture:

**Nimblegen Capture:**

**-   Published POP exp 2007 using TSP gene set,**
**  ****Tested WGA data**** **
**   ****Tested Whole exome chip set**
**   “****Integration and optimization”**

## NimbleGen Capture-454 Pipeline

**	****PRE-CAPTURE LAB:**
- Fragment DNA to 500 bp range by nebulization
- End repair and ligate on NimbleGen linkers (for downstream PCR)
**	****CAPTURE LAB:**
- Hybridize DNA to chips for 3 days at 42o C using a NimbleGen hyb station
- Wash chip stringently in a series of washes
- Elute bound DNA from chip
**	****POST-CAPTURE LAB:**
- Ligation-mediated PCR of pre- and post-capture DNA with NimbleGen linkers
- Q-PCR and compare fold enrichment for control loci pre- and post-capture
- Generate 454 FLX libraries

## NimbleGen System

**  ****Capture Array Specifications:**
**	**Probe lengths 		50-85mer
- 	Features/Array		385,000
- 	Overall Slide Dimensions 	25 x 75mm
- 	Array size 			17.4mm x 13mm
- 	Feature Size 		16μm x 16μm

**12-bay Hybridization Station**

**Elution Station**

## 1000 Genomes Pilot 3

- 101 samples so far
-  Since April 2008, 98 HapMap samples captured on 1000 exomes arrays (2.3 Mb) and corresponding 454 libraries generated
-  42 sequencing runs on 454 ( 41-FLX and 1-XLR)
- 	- 111 Mb/run average (FLX)
- 	- 427 Mb/run (XLR)

## Slide 23

|  | Original Design |  | Rebalanced Design |  |
| --- | --- | --- | --- | --- |
|  | FLX | XLR | FLX | XLR |
| Total Reads Aligned: | 86.23% | 87.01% | 88.72% | Ongoing expt. |
| Aligned Reads On-Buffer: | 11.37% | 5.78% | 15.91% |  |
| Aligned Reads On-Target: | 49.87% | 64.83% | 
58.56%
 |  |
| Reads that hit target or buffer: | 61.24% | 70.61% | 74.47% |  |
| Targets Hit: | 97.54%	 | 98.24% | 99.68% |  |
| Bases with 5+ coverage: | 70.00% | 87.11% | 
90.95%
 | Projected >95% |
| Bases with 10+ coverage:
 | 50.52% | 79.99% | 
74.80%
 | Projected >90% |

## HapMap Concordance

- 				      **FLX-NA12044**	    **XLR-NA12044**
- Coverage (mean/median)	   19/14X	          63/43X
- No cov. Homozygous SNPs	    23 (5%)		13 (3%)
- Homozygous SNP Hits		   441 **(93%)**	          453 **(96%)**
- Homozygous SNP Miss		      9  (2%)		 7 (1.5%)
- Total Homozygous SNPs	   473		           473
- No cov. Heterozygous SNPs	    38 (6%)		17 (3%)
- Heterozygous SNP Hits	   539 **(88%)****	**          573 **(94%)**
- Heterozygous SNP Miss	    35 (6%)		22 (3.5%)
- Total Heterozygous SNPs	    612		           612

## SNP Calling

## Performance Parameters 

- Fold coverage
- Percentage of reads that align
- Percentage of reads that hit the target
- Percentage of reads with unique start points
- Verification of SNPs and percentage of false negatives and false positives
- 	- compare to HapMap genotypes if available
- 	- compare to dbSNP
- 	- compare to other sequence information (example ENCODE regions by Sanger sequencing)
- 	- compare to sequencing results obtained on an alternate next generation platforms (SOLiD) for Pilot 1 and 2 samples
- Ideal: 90% of bases with 10X coverage

## R & D and Other Capture Projects

- 	- Directly add on 454 adaptors to pre-capture library to obviate the need to construct a post-capture 454 library
- 	- Decrease the amount of input DNA for pre-capture (2X) and hybs (3.3X) = 6.6X fold reduction (20 ug down to 3 ug input DNA)
- 	- Comparison of genomic DNA versus WGA
- 	- In–house wash-elution station
- 	- Alternate chip substrate surfaces in collaboration with NimbleGen
- 	- Probe optimization experiments in collaboration with NimbleGen
- 	- HD2 arrays (2.1 million features)

## Ovarian Plan:

- 24 samples on Gene list A
- Test new methods on Gene list A as well
- Use validation to switch to new methods
- Jan 1 09 target for full transition

## Summary

- 	- Whole genome sequencing tested with 454
- 	- Whole GBM plan uses 454 + SOLiD
- 	- Ovarian phased Sanger – Capture (01/09)
- 	- Capture methods ‘more optimized’

## Acknowlegdements

**HGSC**
- Donna Muzny
- Aniko Sabo
- Maggie Morgan
- SDavid Wheeler
- George Weissenberger
- Qing Fu
- Sandy Lee
- Matthew Bainbridge
- Jeffrey Reid
- David Wheeler
- Christie Kovar
- Huyen Dinh

**NimbleGen**
**Thomas Albert**
- Xinmin Zhang
- Jacob Kitzman
- Dan Burgess
- Jeffrey Jeddeloh
- Mark D’Ascenzo
**454**
**Michael Egholm**
- Jonathan Rothberg