Question: 【tophat】 RNA-seq mapping reference genome
0
gravatar for bio_zhangxl
4.4 years ago by
bio_zhangxl10
China
bio_zhangxl10 wrote:

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).

 

 

tophat output • 2.9k views
ADD COMMENTlink modified 3.2 years ago by herman.pappoe.4510 • written 4.4 years ago by bio_zhangxl10
1
gravatar for bio_zhangxl
4.4 years ago by
bio_zhangxl10
China
bio_zhangxl10 wrote:

Now I know the reason now .

the memory is not enough,and the file can not be writted into the folder. 

 

ADD COMMENTlink written 4.4 years ago by bio_zhangxl10
0
gravatar for geek_y
4.4 years ago by
geek_y9.7k
Barcelona/CRG/London/Imperial
geek_y9.7k wrote:

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.

ADD COMMENTlink written 4.4 years ago by geek_y9.7k
0
gravatar for bio_zhangxl
4.4 years ago by
bio_zhangxl10
China
bio_zhangxl10 wrote:

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 ?

ADD COMMENTlink written 4.4 years ago by bio_zhangxl10

What was the error you faced ?

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

ADD REPLYlink written 4.4 years ago by geek_y9.7k
0
gravatar for bio_zhangxl
4.4 years ago by
bio_zhangxl10
China
bio_zhangxl10 wrote:

what does ' you can get it from the kit' mean ? where can get it?

ADD COMMENTlink written 4.4 years ago by bio_zhangxl10
0
gravatar for bio_zhangxl
4.4 years ago by
bio_zhangxl10
China
bio_zhangxl10 wrote:

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

 

ADD COMMENTlink written 4.4 years ago by bio_zhangxl10
0
gravatar for herman.pappoe.45
3.2 years ago by
United States
herman.pappoe.4510 wrote:

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

ADD COMMENTlink modified 3.2 years ago • written 3.2 years ago by herman.pappoe.4510
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