Script error for TopHat & Bowtie 2 for paired end and splice variant detection but?

[Richard Barker]

I have designed this script to run Bowtie2 on paired end fastq data files for a spliceoform investigation. However, it fails as it can't find the -1 input file, this is very strange as the name is correct...
Please help as i'm not sure what to try?

tophat -o 9-0GS_bowtie2_for_splicegrapher -j TAIR10_GFF3_genes.gff --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 TAIR10_chr_all.bt2 -1 9-0GS_AGTCAA_run298_trimmed_1.fastq -2 9-0GS_AGTCAA_run298_trimmed_2.fastq


[2012-08-09 15:17:04] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2012-08-09 15:17:04] Checking for Bowtie
Bowtie version: 2.0.0.7
[2012-08-09 15:17:04] Checking for Samtools
Samtools version: 0.1.18.0
[2012-08-09 15:17:04] Checking for Bowtie index files
[2012-08-09 15:17:04] Checking for reference FASTA file
Warning: Could not find FASTA file TAIR10_chr_all.bt2.fa
[2012-08-09 15:17:04] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bin/bowtie2-inspect TAIR10_chr_all.bt2 > 9-0GS_bowtie2_for_splicegrapher/tmp/TAIR10_chr_all.bt2.fa
[2012-08-09 15:17:09] Generating SAM header for TAIR10_chr_all.bt2
Traceback (most recent call last):
File "/usr/local/bin/tophat", line 3948, in <module>
sys.exit(main())
File "/usr/local/bin/tophat", line 3808, in main
params.read_params = check_reads_format(params, reads_list)
File "/usr/local/bin/tophat", line 1725, in check_reads_format
zf = ZReader(f_name, params)
File "/usr/local/bin/tophat", line 1678, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: '-1'

But the directory is there, with the correct name, i've also tried with the whole root directory and got the same message.

Any ideas?

[kmcarr]

tophat -o 9-0GS_bowtie2_for_splicegrapher -j TAIR10_GFF3_genes.gff --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 TAIR10_chr_all.bt2 -1 9-0GS_AGTCAA_run298_trimmed_1.fastq -2 9-0GS_AGTCAA_run298_trimmed_2.fastq

[snip]...
Warning: Could not find FASTA file TAIR10_chr_all.bt2.fa
...[snip]
Any ideas?

Richard,

It's not complaining about not being able to find the read file; it can not find the fasta file associated with your bowtie index. This is because you passed the wrong name for the index file to bowtie. Do not include the .bt2 suffix in the name of the index. Also you need to use '-x' before the index name in your command line. You should specify the index as

-x TAIR10_chr_all

And make sure that your BOWTIE_INDEXES environment variable is properly set so that it can find the index.

ETA: Do not use -x! The index name and the read file names do not take option letters.

[Richard Barker]

Thanks for the swift response.

I replaced TAIR10_chr_all.bt2 with -x TAIR10_chr_all
and that solved the warning about not finding the FASTA file. Thanks!

But now it just says
tophat: option -1 not recognized

even when i give it the specific fastq file location?

richard@ubuntu:~/RNA_seq_analysis/paired_end$ tophat -o 9-0GS_bowtie2_for_splicegrapher --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 -x TAIR10_chr_all -1 /home/richard/RNA_seq_analysis/paired_end/9-0GS_AGTCAA_run298_trimmed_1.fastq -2 /home/richard/RNA_seq_analysis/paired_end/9-0GS_AGTCAA_run298_trimmed_2.fastq
tophat: option -1 not recognized

[kmcarr]

Thanks for the swift response.

I replaced TAIR10_chr_all.bt2 with -x TAIR10_chr_all
and that solved the warning about not finding the FASTA file. Thanks!

But now it just says
tophat: option -1 not recognized

even when i give it the specific fastq file location?

richard@ubuntu:~/RNA_seq_analysis/paired_end$ tophat -o 9-0GS_bowtie2_for_splicegrapher --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 -x TAIR10_chr_all -1 /home/richard/RNA_seq_analysis/paired_end/9-0GS_AGTCAA_run298_trimmed_1.fastq -2 /home/richard/RNA_seq_analysis/paired_end/9-0GS_AGTCAA_run298_trimmed_2.fastq
tophat: option -1 not recognized

Richard,

What version of TopHat are you running? When I saw in your original post mention of Bowtie2 I assumed you were running TopHat2. To determine which version just run the command:

tophat -v

and report the output.

[Richard Barker]

used tophat -v and says I'm running TopHat v2.0.4

[kmcarr]

used tophat -v and says I'm running TopHat v2.0.4

Ah...now I see the problem. You are running tophat but trying to pass command line options as if were bowtie. You do not use the -1 or -2 for tophat (you also should not use -x for the index, that was my mistake, sorry).

Have a good look at the documentation for TopHat2. No option identifiers are used for the index or read files. They are the last three parameters passed to tophat, in the order shown. Your command should be in the format:

# tophat [options for tophat] <bowtie_index_name> <read1_file> <read2_file>

[Richard Barker]

Thanks kmcarr, it's running now (if interested the final script is below) and was designed for out puts that can be used by SpliceGrapher (Roger et al 2012 http://splicegrapher.sourceforge.net/).

The script below has a lot of advance options that were recommended by the creators of SpliceGrapher inorder to optimise splice varient detection. Will the outputs also be suitable for cuffmerg and cuffdiff analysis?

tophat -o 9-0GS_bowtie2_for_splicegrapher --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 TAIR10_chr_all 9-0GS_AGTCAA_run298_trimmed_1.fastq 9-0GS_AGTCAA_run298_trimmed_2.fastq

[Richard Barker]

It worked for the first 2 files but when i went to run the second sample i got the following error message, i'm really not suer what to do?

richard@ubuntu:~/RNA_seq_analysis/run299_paired_end_for_BowTie_2$ tophat -o 10-0MS_tophat_owtie2_for_splicegrapher --no-convert-bam -a 10 -g 1 -F 0 -p 6 --segment-length=28 --segment-mismatches=0 -i 40 -I 5000 --min-coverage-intron=40 --max-coverage-intron=5000 --min-segment-intron=40 --max-segment-intron=5000 TAIR10_chr_all.bt2 10-0MS_AGTTCC_L002_run298_R1_trimmed.fastq 10-0MS_AGTTCC_L002_run298_R2_trimmed.fastq

[2012-08-12 14:40:38] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2012-08-12 14:40:38] Checking for Bowtie
Bowtie version: 2.0.0.7
[2012-08-12 14:40:38] Checking for Samtools
Samtools version: 0.1.18.0
[2012-08-12 14:40:38] Checking for Bowtie index files
[2012-08-12 14:40:38] Checking for reference FASTA file
Warning: Could not find FASTA file TAIR10_chr_all.bt2.fa
[2012-08-12 14:40:38] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bin/bowtie2-inspect TAIR10_chr_all.bt2 > 10-0MS_tophat_owtie2_for_splicegrapher/tmp/TAIR10_chr_all.bt2.fa
[2012-08-12 14:40:43] Generating SAM header for TAIR10_chr_all.bt2
format: fastq
quality scale: phred33 (default)
[2012-08-12 14:40:43] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: qual length (121) differs from seq length (87) for fastq record !

[kmcarr]

Richard,

This is exactly the same error as your original post. Check the solution up-thread (but ignore the bit about adding -x).