Concatenating fastq files for better assembly
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8 months ago
shail.nair05 ▴ 20

I have sequenced metatranscriptomic sample with technical replicate (same extrected mRNA was sequenced twice). Now, whem i assemble them individually with rnaspades, overall i get similiar results between technical duplicate, but there are a few differecnces in expression of a few metabolic transcripts (like in one sample of same set does not show thisparticular transcript of interest while in other it does).

Can i Concatenate the cleaned fastq of technical replicates (as they are from the same RNA source and sequenced using same library) and re-run the assembly. If so, how do i further calculate the raw read abundance? Do i need to take average or total raw read number (which does not make sense as i will be Concatenating the samples)?

genomics assembly sequencing RNA-Seq • 424 views
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You are going to need to try and see if that makes a difference. If your metatranscriptome is from bacteria then rnaSPAdes may not be making any difference. Some limited testing I had done seemed to indicate that all three modes (normal, meta and RNA) SPAdes produced more or less similar results.

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I have not tested the other spades method. but rnaspades gave me a better assembly than megahit. Similarly, there were a few differences in transcripts between technical replicates assembled via rnaspades. After Concatenating the technical replicates i i got the transcripts of interests (which earlier was not shown in one of the replicate).

Now to get the raw read counts of the transcript, do i map the transcript back to not Concatenated fasrq files or to Concatenated fastq files (which will show higher abundance of overall transcripts as the fastq files are Concatenated).

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