Question: Detection of transcripts from mRNA and sRNA
0
gravatar for kamel
7 months ago by
kamel0
kamel0 wrote:

Hello,

I have two fastq file of mRNA and sRNA sequenced from a patient infected with a pathogenic bacterium. My goal is to identify the transcripts of both the patient and the bacteria.

 Is it logical to make a mapping with two reference sequences (one for the huamin genome and the other for the bacteria ??

Could you offer me a workflow to detect the trancrits??

Thank you in advance

rna-seq alignment assembly • 467 views
ADD COMMENTlink modified 7 months ago by h.mon19k • written 7 months ago by kamel0
3
gravatar for Devon Ryan
7 months ago by
Devon Ryan84k
Freiburg, Germany
Devon Ryan84k wrote:

The ideal procedure would be to concatenate the two genomes and align to that.

ADD COMMENTlink written 7 months ago by Devon Ryan84k

Thank you Devon Ryan for your reply. in this case I will get a single alignment file but how can I count the transcripts of both organisms by a single file. I can not find a good methodology for my case

ADD REPLYlink written 7 months ago by kamel0
2

They'll have quite different chromosome names, so either run featureCounts twice (once with each GTF files), or concatenate the GTF files and run featureCounts using that. I would suggest running featureCounts twice, since I expect you'll want to analyse the two organisms separately anyway.

ADD REPLYlink written 7 months ago by Devon Ryan84k

thank you Devon for your answer, so I will run twice featureCounts to analyze the two organizations separately. I just come back on the concatenation of the two references genomes, do you find this useful command : cat reference 1.fasta reference2.fasta> all_genomes.fasta (because for each reference sequence has different chromosomes)

ADD REPLYlink written 7 months ago by kamel0
1

Yes, just cat the files together, exactly like that.

ADD REPLYlink written 7 months ago by Devon Ryan84k

Hi Devon, I used featureocunts for quantization but its output is complicated. do you know how I can Generate an account matrix with featureCounts

ADD REPLYlink written 5 months ago by kamel0

Another question Plz. For sRNA mapping, I find somebody who selects reads from 18-30 and others from 18-26 before mapping. I want to know what size to select??

ADD REPLYlink written 6 months ago by kamel0
1

It depends on the type of RNA you're interested in. You size select for those types.

ADD REPLYlink written 6 months ago by Devon Ryan84k
1
gravatar for h.mon
7 months ago by
h.mon19k
Brazil
h.mon19k wrote:

Yes, you can build a new reference combining both human and bacterial references. Another option is to use some tool to split the reads between the correct genomes. There are other tools, but I can recommend bbsplit.sh from the BBTools / BBMap package.

ADD COMMENTlink written 7 months ago by h.mon19k

Thanks h.mon for your help this is a tool I'm going to use, but I saw that it gives both .bam file for each alignment with reference, and that's the question I asked Devon. how can I count the transcripts for both organisms at once.

ADD REPLYlink written 7 months ago by kamel0
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