ribosome profiling problems

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4.8 years ago

blur ▴ 280

Hi,

I am working on ribosome profiling - I downloaded, trimmed and aligned reads to rRNA using bowtie2, then took the unmapped reads and aligned them to the ucsc_hg19 reference with the hg19 gtf using TopHat2.

The thing is - there is a very low alignment rate (only 3498 reads were aligned). Which makes me think I did something wrong... I did a check with BWA mem to see if the rates are better - and they are much much higher. The cmd. is:

/nadata/software/tophat-2.0.12.Linux_x86_64/tophat2 -G ucsc_hg19.gtf -o ./ribo_bam -M -p 3 --no-novel-juncs -g 5 ./ucsc_hg19 4TopHat_reads.fastq

Can anyone tell me what I'm missing?

ribosome profiling • 1.2k views

ADD COMMENT • link 4.8 years ago by blur ▴ 280

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There is no reason to use tophat2 in this day and age, use STAR or another more modern aligner. Your alignment rates will likely be MUCH higher.

ADD REPLY • link 4.8 years ago by Devon Ryan 104k

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I am also planning to use HISAT, but was specifically asked to do TopHat2 as well (really not my choice) so that I could compare to a published dataset...

ADD REPLY • link 4.8 years ago by blur ▴ 280

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Either ignore requests like that or learn to say "no" to them, you're probably not getting paid to waste your and their time. It's better to reprocess published datasets.