alignment of sequencing data to the tRNA transcriptome

Question

[alirezamomeni707]

I have ribosome sequencing data and want to align it to the tRNA (not mRNA transcriptome). I have filtered out rRNA, non-coding RNAs and adapter. also I made tRNA indexes using bowtie2 (I have .bt2 files). now I want to align my data to the tRNA transcriptome. I have tried this command but did not give me BAM file. do you know how to solve the problem.

tophat -n 2 -m 1 --segment-length 25 --no-novel-juncs
--no-novel-indels --no-coverage-search -G gencode.v19.tRNAs.gtf -p 1 -o Ctrl_tophat_out tRNAs Ctrl.fastq.gz

[Buffo]

why tophat? you can use bowtie2 for that.

[alirezamomeni707]

I actually tried did not work. maybe my command was wrong. do you have an example?

[Buffo]

You need;

reference.fasta
seqs_to_map.fasta
make reference index;
bowtie2-build reference.fasta ref_index
It produces the index; 6 files .bt2
Then make the mapping;
bowtie2 --np 0 --n-ceil L,0,0.02 --rdg 0,6 --rfg 0,6 --mp 6,2 --score-min L,0,-0.2 -x ref_index -f  seqs_to_map.fasta -S file_out.sam
then sort and create .bam with samtools:

samtools view -bS file_out.sam > file_out_2.bam
samtools sort file_out_2.bam file_out_2.sor
samtools index file_out_2.sor.bam #to load in igv for example

That`s it

[Devon Ryan]

1. Did it produce an error?
2. Why are you using tophat (it's a deprecated tool)?

[alirezamomeni707]

what tool do you suggest? actually I need to get bam file.

[Devon Ryan]

If for some reason you actually need a splice-aware aligner (unlikely) then you can just use bowtie2 directly.

[alirezamomeni707]

here is the error I got after running this command:

Error: Couldn't build bowtie index with err = 1

[st.ph.n]

Edit to show the command you used.