Question: QC and biological repeats of small RNA sequencing data
gravatar for kevolinchen
4.2 years ago by
kevolinchen30 wrote:

When using fastqc to assess the quality of small RNA-seq data, what is a good results? (It's seems that the quality is not good for all the data)

How to deal with biological repeats (e.g. 3 fastq files)?

Is it ok to merge them first? If so, how to merge them, any tools? Can I just use

$cat A_1_R1.fastq A_2_R1.fastq A_1_3_R1.fastq > A_merge.fastq?

What is the difference bewteen sRNA-seq and RNA-seq when filtering?

For now I just try to trim the adapter and remove reads form repeat region of genome.

I think really need some tutorial for sRNA-seq anaylsis....

Do any biostarers have some tools or pipelines to share for sRNA-seq analysis? 

Thank you~


ADD COMMENTlink modified 4.1 years ago by Biostar ♦♦ 20 • written 4.2 years ago by kevolinchen30
gravatar for Devon Ryan
4.2 years ago by
Devon Ryan88k
Freiburg, Germany
Devon Ryan88k wrote:

Take all of the warnings/errors that FastQC produces with a very large grain of salt (they're most appropriate for DNA sequencing).

Regarding the biological replicates, keep them separate.

sRNA-seq has a different library prep. than regular RNAseq, where there's a stringent size-selection step in the former (resulting in mostly small (<35bases or so) transcripts being sequenced). Regular RNAseq doesn't normally have a size selection step (excluding the fact that some kits won't efficiently produce transcripts less than ~200 bases), though either poly-A selection or ribo-depletion are common.

For tutorials, search pubmed.

Regarding pipelines, it depends on whether you're mostly interested in miRNAs or another single species or if you want everything in a single go. Most of the published pipelines (e.g., miRdeep2) are targeted toward a single RNA species...though since most people are interested in miRNAs this makes sense.

ADD COMMENTlink written 4.2 years ago by Devon Ryan88k

Thank you. But how to integrate biological replicates? Average after counting or ...?

I am now trying miRdeep2.:)

ADD REPLYlink modified 4.2 years ago • written 4.2 years ago by kevolinchen30

You would use them in the downstream statistics, which would rely on replicates due to using a negative-binomial model.

ADD REPLYlink written 4.2 years ago by Devon Ryan88k

is there any way to view samples together on the same chart/graph in FastQC, or any other fastq QC analysis package?




ADD REPLYlink written 4.2 years ago by matt.arno0
gravatar for Lars
4.2 years ago by
Lars640 wrote:

You could give DARIO a try:

ADD COMMENTlink written 4.2 years ago by Lars640

Thank you, that seems to be a user friendly web tool~  But I am afraid that my data are too big to analysis online (44 samples). So, I'd better find a pipeline or build a pipeline can be run on our server.

ADD REPLYlink written 4.2 years ago by kevolinchen30
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