【tophat】 RNA-seq mapping reference genome

Question

[bio_zhangxl]

Mapping the RNA-seq reads to reference genome:

tophat --GTF Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf Mus_musculus/UCSC/mm9/Sequence/Bowtie2Index/genome SRR1508783_1.fastq SRR1508783_2.fastq

There are only three file in the tophat_out:

logs  prep_reads.info  tmp

I do not know where are the accepted_hits.bam ,junctions.bed, accepted_hits.bam files.

or I use tophat in the wrong way.

I want to do it again, and try to get the parameter -r. But I can not find the fragments length.

Just know this is a pair-end data(101bp,99bp).

[herman.pappoe.45]

Hello I am experiencing the same issue as the OP. I have increased the memory as you have suggested. My command line looks like this:

qsub -cwd \
  -V \
  -l num_proc=8,h_data=32G,h_rt=24:00:00 \
  -N day0_tophat \
  -b y \
  "module load tophat;
   module load bowtie2;
   module load samtools;
   tophat -g 1 -p 6 -o tophat_output0 -G /path/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf /path/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome day0.fastq"

Unfortunately I am still experiencing what the OP was experiencing in terms of output files and lacking accepted_hits.bam files. There are no errors. It is still running and these are my current output results:

genes.1.bt2
genes.2.bt2
genes.3.bt2
genes.4.bt2
genes.bwt.samheader.sam
genes.fa
genes.fa.tlst
genes.juncs
genes.rev.1.bt2
genes.rev.2.bt2
genes.ver
genome_genome.bwt.samheader.sam
left_kept_reads.bam
left_kept_reads.bam.index
left_kept_reads.m2g.bam
left_kept_reads.m2g.bam.index
left_kept_reads.m2g_um.bam
left_kept_reads.m2g_um.bam.index
left_kept_reads.m2g_um.mapped.bam
left_kept_reads.m2g_um.mapped.bam.index
left_kept_reads.m2g_um_seg1.bam
left_kept_reads.m2g_um_seg1.bam.index
left_kept_reads.m2g_um_seg1.fq.z
left_kept_reads.m2g_um_seg1_unmapped.bam
left_kept_reads.m2g_um_seg1_unmapped.bam.index
left_kept_reads.m2g_um_seg2.bam
left_kept_reads.m2g_um_seg2.bam.index
left_kept_reads.m2g_um_seg2.fq.z
left_kept_reads.m2g_um_seg2_unmapped.bam
left_kept_reads.m2g_um_seg2_unmapped.bam.index
left_kept_reads.m2g_um_unmapped.bam
left_kept_reads.m2g_um_unmapped.bam.index
segment.deletions
segment.fusions
segment.insertions
segment.juncs
temp.samheader.sam

What else can I do?

-EDIT Please disregard I found the problem

Answer 1

[bio_zhangxl]

Now I know the reason. The memory is not enough,and the file can not be written into the folder.

Answer 2

[GouthamAtla]

Use tophat2 with following command and post if you see any error/warnings

tophat2 -o tophat_out -p <int> -G genes.gtf genome_index sample1_R1.fq sample1_R2.fq

Regarding the insert size, you can leave it to defaults, but you can get it from the kit they have used to prepare libraries.

[bio_zhangxl]

Thank you so much.

Can you tell me why the first time I get no useful results?

Why did not generate accepted_hits.bam junctions.bed accepted_hits.bam files.

The parameter -o defaults in the tophat_out ,but there are only (logs, prep_reads.info, tmp)

And -r default value=50, seem not precise

What is the function of GTF file? If I want get small RNA (from 25bp to 30bp), not genes, is it necessary to use it?

[GouthamAtla]

What was the error you faced ?

GTF file will be used for constructing transcriptome. Read the top hat paper for detailed steps.

[bio_zhangxl]

I get these files in the tmp folder,but there are not accepted_hits.bam junctions.bed file. There are no errors, I do not know why.

left_kept_reads.m2g_um_seg3.bam
right_kept_reads.bam
right_kept_reads.m2g.bam
left_kept_reads.m2g_um.bam
left_kept_reads.bam
left_kept_reads.m2g_um_seg2.bam
left_kept_reads.m2g_um_seg1.bam
right_kept_reads.m2g_um.bam
right_kept_reads.m2g_um.mapped.bam
left_kept_reads.m2g_um.mapped.bam
left_kept_reads.m2g_um_seg4.bam
genes.1.bt2
genes.2.bt2
genes.3.bt2
genes.4.bt2
genes.bwt.samheader.sam
genes.fa
genes.fa.tlst
genes.juncs
genes.rev.1.bt2
genes.rev.2.bt2
genes.ver
genome_genome.bwt.samheader.sam
left_kept_reads.bam.index
left_kept_reads.m2g.bam
left_kept_reads.m2g.bam.index
left_kept_reads.m2g_um.bam.index
left_kept_reads.m2g_um.mapped.bam.index
left_kept_reads.m2g_um_seg1.bam.index
left_kept_reads.m2g_um_seg1.fq.z
left_kept_reads.m2g_um_seg1_unmapped.bam
left_kept_reads.m2g_um_seg1_unmapped.bam.index
left_kept_reads.m2g_um_seg2.bam.index
left_kept_reads.m2g_um_seg2.fq.z
left_kept_reads.m2g_um_seg2_unmapped.bam
left_kept_reads.m2g_um_seg2_unmapped.bam.index
left_kept_reads.m2g_um_seg3.bam.index
left_kept_reads.m2g_um_seg3.fq.z
left_kept_reads.m2g_um_seg3_unmapped.bam
left_kept_reads.m2g_um_seg3_unmapped.bam.index
left_kept_reads.m2g_um_seg4.bam.index
left_kept_reads.m2g_um_seg4.fq.z
left_kept_reads.m2g_um_seg4_unmapped.bam
left_kept_reads.m2g_um_seg4_unmapped.bam.index
left_kept_reads.m2g_um_unmapped.bam
left_kept_reads.m2g_um_unmapped.bam.index
right_kept_reads.bam.index
right_kept_reads.m2g.bam.index
right_kept_reads.m2g_um.bam.index
right_kept_reads.m2g_um.mapped.bam.index
right_kept_reads.m2g_um_unmapped.bam
right_kept_reads.m2g_um_unmapped.bam.index
temp.samheader.sam

[bio_zhangxl]

What does 'you can get it from the kit' mean? Where can get it?