Question: RNASeq lane effect
gravatar for roncalli
2.8 years ago by
roncalli0 wrote:

Hi All,

I am looking for an answer..Maybe easy for some of you! I have 2 RnaSeq datasets processed in the same way (same individual, same RNA extraction, cDNA prep and sequencing platform) the only difference is that I ran the 2 datasets on 2 separate lanes. The samples have been multiplexed and have a similar sequencing depth (# of reads). What I am trying to do is to run a gene expression analysis on them. Basically I did map both datasets against a reference transcriptome (previously generated) and I am using the raw count as EdgeR data to run the statistics. My question is...How do I account of the lane difference? Is there a way of quantifying it? Or...does the TMM normalization includes the possible lane error?

Thanks for the help!


ADD COMMENTlink modified 2.8 years ago by zzqr40 • written 2.8 years ago by roncalli0

Any particular reason you expect lane differences? I have seen technical replicates on neighbouring lanes and they look identical.

ADD REPLYlink written 2.8 years ago by Satyajeet Khare1.5k

If the same library was run on two lanes then there should be no difference in the data (read numbers may be more or less if the loading concentration was different).

ADD REPLYlink written 2.8 years ago by genomax85k

there might be something off if you multiplex hardly, and the indexes used within the lane do not co-op with each other well

ADD REPLYlink written 2.8 years ago by stolarek.ir650
gravatar for zzqr
2.8 years ago by
zzqr40 wrote:

If there are two different samples ran on two lanes, the seq data could not tell the difference from lane effect. Have technical replicates might be a good idea to account of the lane difference. The TMM normalization estimates scale factors between samples. It could not distinguish the effect bewteen sample diffence and lane difference.

ADD COMMENTlink written 2.8 years ago by zzqr40
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