I got a fastq files from core and they loaded same library on two different lanes. I read the posts of people that deal with this problem and they recommend to merge these file after converting to bam. My question is next, why I cannot merge these files using tophat into accepted_hits.bam by using these files like technical replicates? What will be the difference between these two methods. I plan to use cufflinks for the analysis of the DE. Thank you for the help.
2 answers
I assume you mean two different lanes on the sequencer rather than lines.
You can either merge the fastq files or merge the bam files, which should lead to the same final outcome. Using those separately as technical replicate probably has probably limited added value.
Note: Tophat-Cufflinks is no longer the recommended RNA-seq processing pipeline. A new protocol is described here: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown, but there are many more alternatives.
Thank you. I know that I can merge either fastq or bam files, however I do not understand why I cannot use tophat to merge these fastq file into single accepted_hits.bam file?
So you mean listing all fastq files per read direction (comma separated) as input to Tophat? That would also be possible, and I assume equivalent to the other options. I see you suggested that in your initial post, although I'm unsure what you mean with "like technical replicates" (because in this case, they're not used as replicates).
Yes you are right I want to use next tophat command to merge these files
tophat -G genes.gtf -p 8 -o Output genome lane1_1.fastq,lane2_1.fastq
and use output bam file for subsequent analysis. Is it correct?
Without looking in the manual, the command looks okay. But you shouldn't use tophat as said before.
Could you please explain, why this is not good idea? What will be difference between these two approaches (merging fastq file using cat and bam files using samtools and merging these file using tophat)? Thank you
There is nothing wrong with the approach of merging the fastqfiles. Just don't use tophat. It's deprecated/low maintenance.
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Thank you very much for help.