WGS library gen gone wrong?

Ggnome · 09-30-2020, 11:25 AM

Hi, I'm doing a WGS sequencing project. The original samples provided were whole human blood in EDTA. After DNA extraction and library gen with the TruSeq DNA PCR-Free protocol w/ 350 bp insert, I got the following results on Tapestation (gel and histogram of one of the 5 libraries, which looks similar to them all):

https://imgur.com/a/T0CJLcS

The result looks a lot different from the figures in the Illumina Library QC whitepaper, so I was wondering if anyone knows if it's a good idea to proceed to sequencing now with this result? If not, any ideas on what went wrong?

I'm concerned because I need to save project budget for the sequencing itself, so I'd rather re-do the DNA extract and library gen than sequence bad libraries and get bad data.

Thanks in advance!

jdk787 · 10-01-2020, 05:29 AM

As long as the qPCR results are good those libraries should be fine to sequence.

luc · 10-01-2020, 11:09 AM

You might want to have a look at this FAQ:
https://dnatech.genomecenter.ucdavis...ree-libraries/

Ggnome · 10-01-2020, 04:32 PM

Thanks so much for your replies jdk787 and luc! This UC Davis Q&A is very helpful.

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09-30-2020, 11:25 AM	#1
Ggnome Junior Member Location: San Diego Join Date: Jul 2020 Posts: 2	WGS library gen gone wrong? Hi, I'm doing a WGS sequencing project. The original samples provided were whole human blood in EDTA. After DNA extraction and library gen with the TruSeq DNA PCR-Free protocol w/ 350 bp insert, I got the following results on Tapestation (gel and histogram of one of the 5 libraries, which looks similar to them all): https://imgur.com/a/T0CJLcS The result looks a lot different from the figures in the Illumina Library QC whitepaper, so I was wondering if anyone knows if it's a good idea to proceed to sequencing now with this result? If not, any ideas on what went wrong? I'm concerned because I need to save project budget for the sequencing itself, so I'd rather re-do the DNA extract and library gen than sequence bad libraries and get bad data. Thanks in advance!

10-01-2020, 05:29 AM	#2
jdk787 josh kinman Location: Austin Join Date: Apr 2014 Posts: 71	As long as the qPCR results are good those libraries should be fine to sequence. __________________ Josh Kinman

10-01-2020, 11:09 AM	#3
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	You might want to have a look at this FAQ: https://dnatech.genomecenter.ucdavis...ree-libraries/

10-01-2020, 04:32 PM	#4
Ggnome Junior Member Location: San Diego Join Date: Jul 2020 Posts: 2	Thanks so much for your replies jdk787 and luc! This UC Davis Q&A is very helpful.