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Old 03-06-2011, 02:25 PM   #1
Ulli
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Unhappy Exome Sequencing with NimbleGen SeqCap EZ v2

Hi all!

We used NimblGen's SeqCap EZ Human Exome Library v2.0 followed by paired-end sequencing on the Illumina GAIIx for exome sequencing. BWA was used for sequence alignment. About 95% of the reads could be aligned to the genome. However, only about 60% of all aligned reads align within the target region. NimbleGen says, that 80% on target is feasible.

Did anyone reach 80% with this library? Any experiences with this library are welcome. We used the SeqCap v.1 library first and achieved the same percentage on target (60%). So the new version did not improve the results in our case. Merely the target region is larger now.

Best wishes,
Ulli
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Old 03-07-2011, 06:32 AM   #2
upenn_ngs
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what is the % reads on target including +/-100bp? if this metric shows >90% reads on target then i suspect your coverage issue can be resolved by narrowing the gDNA insert size based on your preferred read length. (i.e. ~200bp insert for 2x100bp reads)
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Old 03-07-2011, 07:08 AM   #3
Ulli
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Hi!

I used the "Tiled Region" provided by NimbleGen as .bed file for my calculations.
(http://www.nimblegen.com/products/se...tml#annotation)

When I understand you correctly, it might happen that I have reads with one end of the read on target and the other end off target. In my calculations, such an overlapping paired-end read is counted as one sequence on target and one sequence off target. I will extend the Tiled Region by 300bp and recalculated the % on target. Maybe this will increase the % on target to ~ 80%.

However, I wonder why I obtained the advertised 60% with the version 1 library, even though I used the same strict method to calculate the % on target.

Regards,
Ulli
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Old 03-07-2011, 08:57 AM   #4
upenn_ngs
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could be the probes used to target additional loci in v2 are less stringent, or a difference in sample prep

Last edited by upenn_ngs; 03-07-2011 at 10:10 AM.
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Old 04-25-2011, 06:21 PM   #5
Michael.James.Clark
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My numbers are consistently better than those Nimblegen quotes.

My experience:

At 80M reads, >97% of tiled regions were covered at 10X or higher, and nearly 100% of tiled bases are covered at least once.

Randomly extracting 20M reads from that, >83% of tiled regions are covered at 10X or higher, and almost 97% of tiled regions are covered at least once.

Edit:

Sorry, I think I misunderstood. It looks like your confusion is regarding "off-target" enrichment, not coverage of the tiled intervals. Expect a very large number of alignments to regions immediately adjacent to the tiles. We expect >90% (the number currently quoted by Nimblegen as far as I know) of the tiles to have reads covering them at 10X or higher generally. However, I also find significantly higher coverage outside the tiled intervals than Nimblegen claims. That said, it doesn't negatively impact the exome enrichment and simultaneously yields extra in the introns/UTRs, so I don't see it as a bad thing.
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Last edited by Michael.James.Clark; 04-26-2011 at 11:46 AM.
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