Question: Average insert size for paired end Illumina RNA-seq
1
gravatar for xiefanfang
6.4 years ago by
xiefanfang10
United States
xiefanfang10 wrote:

Does anyone know how to get the average insert size of paired-end RNA seq data (excluding linkers and adapters)? These are Illumina Hiseq2000 data with 2X100 bp run. Thank you!

 

 

sequencing rna-seq next-gen • 7.6k views
ADD COMMENTlink modified 6.4 years ago by st.ph.n2.5k • written 6.4 years ago by xiefanfang10
4
gravatar for Asaf
6.4 years ago by
Asaf8.5k
Israel
Asaf8.5k wrote:

You can't get this data from the sequencing but if prior to sequencing you (or the one that prepared the libraries) used bioanalyzer or other tool to measure the length of the DNA fragments, you can use this data to compute the length of the inserts.

After you map the reads to the genome you can get the length of each fragment from column 10 (TLEN) of the SAM file, just compute the average of the positive values (>0) in this column.
ADD COMMENTlink written 6.4 years ago by Asaf8.5k

For future readers: TLEN is the field number 9, not ten.

ADD REPLYlink written 3.3 years ago by Macspider3.3k
0
gravatar for st.ph.n
6.4 years ago by
st.ph.n2.5k
Philadelphia, PA
st.ph.n2.5k wrote:

A little more lengthy of an option: Post-trimming adaptors/barcodes you can align the reads to a reference, or your own de novo assembly using bwa or bowtie. You can then use picard tools (CollectInsertSizeMeterics), and you will get a nice histogram of the insert sizes in your library.

ADD COMMENTlink modified 15 months ago by _r_am32k • written 6.4 years ago by st.ph.n2.5k
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