Question: RNASEQ preprocessing and insert size
0
gravatar for archie
6 months ago by
archie40
India
archie40 wrote:

Dear all,

I have RNASeq data. I have to estimate insert size for multiple samples. For this, I am using the CollectInsertSizeMetrics picard application. Here I have to provide input of BAM file (reads mapped to reference). I am confused whether I should do preprocessing of data and then estimate insert size or I should directly move with raw data to estimate insert size. please look into following points/conditions....

  1. consider all the reads for mapping and then calculate insert size ?
  2. perform quality filtration,adapter removal, mapping and then estimate the insert size ?
  3. only perform the adapter removal and then estimate the insert size ?

Every step will make the difference. I just want to follow the accurate way to estimate insert size.

Waiting for reply

Thank you in advance

rna-seq • 281 views
ADD COMMENTlink modified 6 months ago by genomax48k • written 6 months ago by archie40

You might want to take a look at this blog: https://mikelove.wordpress.com/2016/09/26/rna-seq-fragment-sequence-bias/ and at the alpine bioconductor package.

ADD REPLYlink written 6 months ago by Sean Davis24k
0
gravatar for Devon Ryan
6 months ago by
Devon Ryan79k
Freiburg, Germany
Devon Ryan79k wrote:

If you're going to filter the data anyway then filter it before performing the estimation. You care about the insert size of the data you'll actually use. Do track how much data is filtered, though, since that will also indicate whether there was an issue during library prep.

BTW, you might need to align against the transcriptome to get an accurate number.

ADD COMMENTlink written 6 months ago by Devon Ryan79k
0
gravatar for genomax
6 months ago by
genomax48k
United States
genomax48k wrote:

If all you are interested in is finding insert size then use BBMap suite like this. There are two ways you can estimate the insert size.

ADD COMMENTlink written 6 months ago by genomax48k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1146 users visited in the last hour