SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Pooling multiplexed libraries after ligation, before PCR ScottC Illumina/Solexa 5 05-07-2016 01:10 PM
Equimolar pooling of hundreds of libraries for sequencing nucacidhunter Sample Prep / Library Generation 0 05-08-2014 06:16 AM
Pooling of Nextera and NexteraXT libraries exo Sample Prep / Library Generation 9 02-21-2014 03:46 AM
Pooling and diluting Nextera libraries kmkocot General 0 09-17-2013 06:00 AM
Indexed libraries best pooling strategy spdinesh Sample Prep / Library Generation 3 02-07-2012 11:21 PM

Reply
 
Thread Tools
Old 04-10-2015, 01:48 AM   #1
jg3197
Junior Member
 
Location: Germany

Join Date: Feb 2014
Posts: 4
Default Pooling reads from different libraries

Hi all,

I have a library I have been given to work with but am having trouble working out if my approach is legitimate. I have 3 libraries each containing 2 tissues from males, and another three libraries for females, one containing both tissues, and the other two with the tissues samples separately, e.g.

Sample 1 - Male - Tissue 1+2
Sample 2 - Male - Tissue 1+2
Sample 3 - Male - Tissue 1+2
Sample 4 - Female - Tissue 1+2
Sample 5 - Female - Tissue 1
Sample 6 - Female - Tissue 2

Is it legitimate to combine sample 5 and 6 to have two replicates for females to compare against the males? I wondered if this would cause problems for fpkms (which are quite sensitive to library sizes) and/or counts as the combined sample would then contain very wildly different numbers of reads?

Many thanks,

Jen
jg3197 is offline   Reply With Quote
Old 05-18-2015, 02:19 AM   #2
jg3197
Junior Member
 
Location: Germany

Join Date: Feb 2014
Posts: 4
Default

I am still struggling with this problem and wondered if anyone had a similar experience? To clarify my problem further, the libraries detailed above were already sequenced before I got the data to work on, so I had no part in the experimental design, which I know is not ideal. So I actually have fastq reads. So, is it ok to combine two fastq files (for samples 5+6) to get one set of reads for tissues 1+2 which will be approximately double the sequence depth of all other tissue samples? The samples were treated in exactly the same way, except that they were separated before libraries were generated.

I think it is ok, but i'm not sure if there's something I have overlooked.

I hope somebody can help me! Thanks.
jg3197 is offline   Reply With Quote
Old 05-18-2015, 08:34 AM   #3
fanli
Senior Member
 
Location: California

Join Date: Jul 2014
Posts: 198
Default

Are the 3 male samples biological replicates from 3 individual males? And similarly for the females? If so, then technically you'd be pooling variation between two females. Read depth shouldn't be an issue if you normalize anyways.

I'd probably do some quick QC with all 6 samples separately first (e.g. just see where everything falls on a PCA).
fanli is offline   Reply With Quote
Old 05-18-2015, 08:53 AM   #4
jg3197
Junior Member
 
Location: Germany

Join Date: Feb 2014
Posts: 4
Default

Hi Fanli, thanks for your reply!

Yes, the males are all separate individuals. For the females, sample 4 is from one individual, and sample 5 and 6 are different tissues of the same individual. So in total, there are 3 males and 2 females.

I did a PCA, and samples 5 and 6 were quite different from each other, which is what i'd expect. I haven't tried a PCA yet combining 5+6 into one sample yet though.
jg3197 is offline   Reply With Quote
Reply

Tags
pooling samples, rna-seq

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 10:00 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO