Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
why the properly paired results from bwa mem is an odd number Pengfei Liu Bioinformatics 2 08-24-2013 06:26 PM
Odd results with Sickle FASTQ trimming id0 Bioinformatics 0 04-19-2013 09:07 AM
Odd "rooster-comb" BioAnalyzer profile for Mate Pair library pbabb926 Sample Prep / Library Generation 3 10-11-2012 10:06 AM
odd total RNA pattern bioanalyzer eab Sample Prep / Library Generation 18 08-18-2012 04:54 AM
BWA produces odd alignment results dandyrilla Bioinformatics 2 11-27-2011 11:28 PM

Thread Tools
Old 08-21-2015, 08:50 AM   #1
Location: iowa

Join Date: Dec 2007
Posts: 10
Default Odd bioanalyzer results

I had a customer that submitted some antibody libraries which I ran on our MiSeq. The results looked good up until about 100 bases then the Q30 took a nose dive and the %A increased, suggesting that the sequence was reading into the adapter on the other side. The gel image showed a library fragment size of about 600 bp, so I had her run it on the Bioanalyzer (high sensitivity DNA). The Bioanalyzer showed a size of about 200 bp, which would be consistent with the MiSeq's poor results, but also showed a large smear at the upper marker, which doesn't show up on the gel image. Can anyone explain why the bioanayzer and agarose gel analyses would give such different results? I have attached a picture of the gel and a bioanalyzer trace of one of the samples
Attached Images
File Type: jpg IMG_0381.JPG (42.6 KB, 48 views)
Attached Files
File Type: pdf 2100 expert_High Sensitivity DNA Assay_DE23101877_2015-08-20_15-03-10.pdf (252.4 KB, 65 views)
beetle_doc is offline   Reply With Quote
Old 08-21-2015, 10:39 AM   #2
Senior Member
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315

The 200 vs 600 bp result, I would tend to blame on different buffer/salt concentrations in the marker( 1KB ladder -- my favorite) and the samples.

Okay, for the high molecular weight stuff visible on the bioanalyzer chip, but not your gel -- I would have to reach into deep speculation, so be forewarned... How about, that material is genomic DNA, single stranded after many cycles of denaturation during in PCR. Very poor visibility of single stranded DNA with the ethidium bromide in your gel, but a high sensitivity chip will visualize it just fine.

Irritatingly, when I originally asked whether single-stranded DNA/RNA would be visible on a high sensitivity chip, Agilient tech support answer that I should not use it for that purpose. Thanks Agilent.
Later experimentation showed that single stranded molecules were visible and migrated aberrantly with respect to their actual length. See here for more details.
pmiguel is offline   Reply With Quote
Old 08-25-2015, 06:28 AM   #3
Location: iowa

Join Date: Dec 2007
Posts: 10

Thanks for the response. I will pass the information along to my client.
beetle_doc is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 05:45 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO