Question: RNASEQ preprocessing and insert size
0
gravatar for archie
12 months ago by
archie70
India
archie70 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 • 416 views
ADD COMMENTlink modified 12 months ago by genomax58k • written 12 months ago by archie70

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 12 months ago by Sean Davis25k
0
gravatar for Devon Ryan
12 months ago by
Devon Ryan86k
Freiburg, Germany
Devon Ryan86k 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 12 months ago by Devon Ryan86k
0
gravatar for genomax
12 months ago by
genomax58k
United States
genomax58k 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 12 months ago by genomax58k
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