SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
DESeq and technical replicates tamari Bioinformatics 0 10-17-2012 04:52 AM
RNA-seq technical replicates at library construction level obig Bioinformatics 9 08-15-2012 11:43 PM
Read runs and technical replicates scottdaniel RNA Sequencing 3 04-30-2012 11:49 AM
Merging reads mapped on genome and CDS (SOLID data) jmandel Bioinformatics 1 01-04-2012 06:34 AM
technical replicates uncorrelate with each other tujchl Epigenetics 4 05-26-2011 06:16 PM

Reply
 
Thread Tools
Old 04-10-2013, 01:13 AM   #1
Fernas
Member
 
Location: Middle East

Join Date: Apr 2013
Posts: 74
Default Merging Mapped Reads from Technical Replicates

Dear All,

I am working to analyze (Illumina 2.0 RNASeq paired-end reads) of length 100bp. these reads belong to 10 biological replicates (different samples of the same species) and each sample was sequenced twice (2 technical replicates). So, in total I have the reads from 20 libraries.

My purpose is to study the differences between biological replicates (samples) and I want to get rid of technical replicates. The option of pooling reads from technical replicates is not desired because if an artifact read occur in one replicate will not be detected.

Consequently, I mapped each library (of the 20 libraries) using tophat 2.0.

Now, I want to do the following:
For each sample, I want to go through all the mapped reads (in accepted_hits.bam files) in both replicates and select reads the common reads in both libraries. The purpose of this step is to remove the effect of artifact reads and to select reads that are verified in 2 technical replicates for each samples. Any idea how to do that?
Fernas is offline   Reply With Quote
Old 04-10-2013, 06:06 AM   #2
Fernas
Member
 
Location: Middle East

Join Date: Apr 2013
Posts: 74
Default

Can I run (samtools mpileup) and from the outputs select minimum read depth at each position?
Fernas is offline   Reply With Quote
Old 04-10-2013, 02:48 PM   #3
shi
Wei Shi
 
Location: Australia

Join Date: Feb 2010
Posts: 235
Default

Quote:
Originally Posted by Fernas View Post
Dear All,

I am working to analyze (Illumina 2.0 RNASeq paired-end reads) of length 100bp. these reads belong to 10 biological replicates (different samples of the same species) and each sample was sequenced twice (2 technical replicates). So, in total I have the reads from 20 libraries.

My purpose is to study the differences between biological replicates (samples) and I want to get rid of technical replicates. The option of pooling reads from technical replicates is not desired because if an artifact read occur in one replicate will not be detected.

Consequently, I mapped each library (of the 20 libraries) using tophat 2.0.

Now, I want to do the following:
For each sample, I want to go through all the mapped reads (in accepted_hits.bam files) in both replicates and select reads the common reads in both libraries. The purpose of this step is to remove the effect of artifact reads and to select reads that are verified in 2 technical replicates for each samples. Any idea how to do that?
I dont think you should do this. Due to the randomness in the fragmentation step in sample prep, there wont be a large overlap in read sequences between the technical replicates. You should instead combine them rather than get the intersection of them.
shi is offline   Reply With Quote
Reply

Tags
combine mapped reads, technical replicates, tophat

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 01:27 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO