Tophat output problem

Question

[amitunited0532]

Please help me to sort out this problem. Why showing 0.0% overall read mapping rate????

My result given below:

Reads: Input : 238477 Mapped : 2 ( 0.0% of input) 0.0% overall read mapping rate.

[Joe]

Well, your first 2 problems are: 1) this is not a “tool” post, and 2) don’t use tophat

[JJ]

use STAR or HISAT2. Tophat is out of date.

[WouterDeCoster]

I have changed this thread from a "Tool" to a "Question".

[cpad0112]

Post your code to understand what is going on.

[amitunited0532]

tophat -G '/home/amit/Desktop/ngs new/archive-2010-09-27-22-25-17/genes.gtf' -o tophat16_2 bowtie2index SRR01545.fastq

[cpad0112]

1. Is your fastq full fastq or part fastq? Reason is that this is a transcriptome assembly. If your fastq is part fastq, probably reads are in non coding regions
2. Does your GTF file genome build match with reference genome? For eg. If your reference (fasta file) is hg19 and your annotations are recent (hg38). Annotations (CDS, Exon etc) may not match. Does chromosome ID match between reference fa and gtf?
3. Is the index file correct? fastq file and index should belong same organism
4. Is there reference file (fasta or fa file - genome.fa or hg19.fa or hg38.fa) in the same directory as index (bowtie2index folder)? Btw, please don't use numbers in directory names. One is supposed to supply directory path for index.

5. Try to use tophat2 instead of tophat if tophat usage is necessary.

   $ tophat2 -o tophat16_2 -G /home/amit/Desktop/ngs new/archive-2010-09-27-22-25-17/genes.gtf bowtie2index .SRR01545.fastq

[amitunited0532]

Thank you for giving valuable time.

1. Fastq file is full
2. I have downloaded Homosapiens_UCSC_hg19.tar.gz (Approx 45.5 GB ). All files obtained after extract.
3. Index and fastq file belong to same Organism (Homo sapiens)
4. I have kept all file in one folder.
5. I will try tophat2 instead tophat.

Thank you

[cpad0112]

To my understanding, code seems to be fine considering above. I guess you have correct files in place and your GTF file annotations from hg19. GTF need not be in quotes.

Independently, take 5 or more reads (sequences) and blast against human genome. Check where they align. Sample your fastq ( some thing like 10%) and try to align instead of entire fastq. See if that works with tophat2.