Unable to run Tophat2

[daanum]

Hi,

I am unable to run Tophat2 as I get an error.

Here is the command I run: tophat2 -p 5 -r 62 –library-type fr-firststrand -G /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/gene.gtf –o /home/jmotwani/RNASeq/Alignment_Tophat2 --BOWTIE2_INDEXES /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/ C95VLANXX-2046D-01-01-01_L003_R1_Trimmed.fastq C95VLANXX-2046D-01-01-01_L003_R2_Trimmed.fastq

I get the following error:


[2016-05-22 22:20:05] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2016-05-22 22:20:05] Checking for Bowtie
Bowtie version: 2.2.9.0
[2016-05-22 22:20:05] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (–library-type.*.bt2l)


The indexed genome was downloaded from Illumina iGenomes page. Do I have to build it after downloading it? I downloaded the genome, gtf file, and indexed files and gave the path of those files in the command above.

Could anyone please comment or advise on this.

Thanks for your time.

Regards, J

[GenoMax]

You need to provide a basename for the index files (which in this case should be genome). So the genome index file path becomes

/home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome

You don't need to include --BOWTIE2_INDEXES. That is a shell variable you could set beforehand.

[daanum]

Thanks Genomax.

I removed the BOWTIE2_INDEXES option and gave the path as suggested above but I still get the same error.

I am wondering if its due to Bowtie2 index version incompatibility. Any comments?

[GenoMax]

Can you show us a listing of

$ ls -lh /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/

[daanum]

The Bowtie2Index folder has following files:

genome.1.bt2
genome.2.bt2
genome.3.bt2
genome.4.bt2
genome.fa
genome.rev.1.bt2
genome.rev.2.bt2
tophat_out

[GenoMax]

Can you try the following? Looks like you had a single - in your library-type option before.

tophat2 -p 5 -r 62 --library-type fr-firststrand -G /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/gene.gtf –o /home/jmotwani/RNASeq/Alignment_Tophat2 /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome C95VLANXX-2046D-01-01-01_L003_R1_Trimmed.fastq C95VLANXX-2046D-01-01-01_L003_R2_Trimmed.fastq

[daanum]

I tried the following:

tophat2 -p 5 -r 62 -–library-type fr-firststrand --GTF /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/gene.gtf –o /home/jmotwani/RNASeq/Alignment_Tophat2 /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome C95VLANXX-2046D-01-01-01_L003_R1_Trimmed.fastq C95VLANXX-2046D-01-01-01_L003_R2_Trimmed.fastq

but now I get a different error:
tophat: option -? not recognized
for detailed help see http://ccb.jhu.edu/software/tophat/manual.shtml

[GenoMax]

Try

tophat2 --num-threads 5 --mate-inner-distance 62 --library-type fr-firststrand --GTF /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/gene.gtf --output-dir /home/jmotwani/RNASeq/Alignment_Tophat2 /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome C95VLANXX-2046D-01-01-01_L003_R1_Trimmed.fastq C95VLANXX-2046D-01-01-01_L003_R2_Trimmed.fastq

There is one last possibility. TopHat may not be liking the - you have in your fastq file names. So if you could change those to "_" if the above does not work.

tophat2 --num-threads 5 --mate-inner-distance 62 --library-type fr-firststrand --GTF /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/gene.gtf --output-dir /home/jmotwani/RNASeq/Alignment_Tophat2 /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome C95VLANXX_2046D_01_01_01_L003_R1_Trimmed.fastq C95VLANXX_2046D_01_01_01_L003_R2_Trimmed.fastq

[daanum]

Thank you. The command worked this time but partly
Command :

tophat2 --num-threads 5 --mate-inner-dist 62 --library-type fr-firststrand --GTF /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf --output-dir /home/jmotwani/RNASeq/Alignment_Tophat2 /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome read1.fastq read2.fastq

Output:


[2016-05-23 22:07:53] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2016-05-23 22:07:53] Checking for Bowtie
Bowtie version: 2.2.9.0
[2016-05-23 22:07:53] Checking for Bowtie index files (genome)..
[2016-05-23 22:07:53] Checking for reference FASTA file
[2016-05-23 22:07:53] Generating SAM header for /home/jmotwani/mydata/Genomes/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome
[2016-05-23 22:07:55] Reading known junctions from GTF file
[2016-05-23 22:08:28] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: qual length (111) differs from seq length (106) for fastq record !

[GenoMax]

That indicates that there is an error in your reads file. What trimming program did you uses (and was it paired-end aware)?

[daanum]

I used a in-house script (cleanadaptors) to trim the raw fastq files. I run the command to trim the data in the following way:

cleanadaptors -I /home/jmotwani/RNASeq/contam.fa -q 20 -x 25 -F C95VLANXX-2046D-01-01-01_L003_R1.fastq -o C95VLANXX-2046D-01-01-01_L003_R1_Trimmed.fastq -G C95VLANXX-2046D-01-01-01_L003_R2.fastq -O C95VLANXX-2046D-01-01-01_L003_R2_trimmed.fastq

-q is for quality and -x is for min length of the read

[GenoMax]

For that you are going to need to consult the person who wrote the script.

If you can't then I suggest that you use bbduk or trimmomatic or cutadapt (a standard trimming program).

[daanum]

I will be using trimmomatic now,thanks for all the help.