Question: RNA-seq: Should remove duplicates in all samples of same experiment although some do not have technical duplicates?
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gravatar for salamandra
7 days ago by
salamandra130
salamandra130 wrote:

1- The graph of % duplication vs read coverage of RNA-seq sample 1 seems to have technical duplicates, but can we say the same for sample 2 and sample 3?

2- If we remove duplicates in one sample (because the graph % duplication vs read coverage suggests there's technical duplicates) should we remove in all the other samples of the same experiment although those samples do not seem to have technical duplicates? Or is it 'unfair' to compare samples with/without duplicates?

3- In the graphs above, what should be the values (high/low) of Int, SI, vertical green line and vertical red line for the sample to have technical duplicates?

ADD COMMENTlink modified 7 days ago • written 7 days ago by salamandra130
1

The question of duplicate removal in RNA-seq has been extensively discussed here multiple times. The short answer is: No, do not remove any duplicates. For the long answer, please use the search function and scan the answers.

ADD REPLYlink written 7 days ago by ATpoint5.5k

Be very careful about messing with "duplicates". Looks like you don't want to heed the notes from last thread mRNA-seq quality report (fastQC): Does it mean samples have adapters and should remove duplicates? .

Unless you had UMI (unique molecular indexes that label each original RNA fragment) it is going to be impossible to determine if you really have PCR duplication.

ADD REPLYlink written 7 days ago by genomax51k

yes, but then i read the link you had provided and understood that at least for the samples where the duplication level vs reads per kilobase graph was 'ugly' we could infer most of the duplicates were technical. And as some of my samples had those 'ugly' graphs i had the question whether we could for those samples remove duplicates and still compare them with the 'good' ones or just discard those samples?

ADD REPLYlink written 7 days ago by salamandra130
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