Hi there, I'm an undergrad working as a summer student in a lab and I'm having trouble with analyzing the RNA-seq data generated by Ion Proton.

The pipeline suggested by Life Technologies involves a 2-step alignment, the first of which is done with tophat2, and I'm stuck on this step...

the command suggested by Life Technologies:

tophat2 -p 12 --keep-fasta-order --GTF known_genes.gtf \
hg19_bowtie2_index adaptorTrim.fastq

my command:

tophat2 -p 2 --keep-fasta-order -G ../RNAseq_test/h_sapiens_37_asm.gtf ~/bowtie2-2.2.3/scripts/h_sapiens_37_asm adaptorTrim.fastq

• the h_sapiens_37_asm.gtf file is actually the ncbi 37.2 build

• h_sapiens_37_asm is the name of the bowtie2 index files built from running the make_h_sapiens_ncbi37.sh script

• adaptorTrim.fastq is the Ion Proton WT RNA-seq data downloaded from Ion Community and trimmed with cutadapt

and I got the following error

[2014-06-11 09:37:40] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-11 09:37:40] Checking for Bowtie
          Bowtie version:     2.2.3.0
[2014-06-11 09:37:40] Checking for Samtools
        Samtools version:     0.1.18.0
[2014-06-11 09:37:40] Checking for Bowtie index files (genome)..
[2014-06-11 09:37:40] Checking for reference FASTA file
    Warning: Could not find FASTA file /home/yzc/bowtie2-2.2.3/scripts/h_sapiens_37_asm.fa
[2014-06-11 09:37:40] Reconstituting reference FASTA file from Bowtie index
  Executing: /home/yzc/bowtie2-2.2.3/bowtie2-inspect /home/yzc/bowtie2-2.2.3/scripts/h_sapiens_37_asm > ./tophat_out/tmp/h_sapiens_37_asm.fa
[2014-06-11 09:40:28] Generating SAM header for /home/yzc/bowtie2-2.2.3/scripts/h_sapiens_37_asm
[2014-06-11 09:40:28] Reading known junctions from GTF file
[2014-06-11 09:40:33] Preparing reads
     left reads: min. length=16, max. length=368, 45510880 kept reads (611 discarded)
Warning: short reads (<20bp) will make TopHat quite slow and take large amount of memory because they are likely to be mapped in too many places
[2014-06-11 10:01:03] Building transcriptome data files ./tophat_out/tmp/h_sapiens_37_asm
[2014-06-11 10:01:07] Building Bowtie index from h_sapiens_37_asm.fa
    [FAILED]
Error: Couldn't build bowtie index with err = 1

I have also tried tophat2 with the same datasets but without applying the gtf file, which resulted in the following error:

Warning: short reads (<20bp) will make TopHat quite slow and take large amount of memory because they are likely to be mapped in too many places
[2014-06-10 15:13:37] Mapping left_kept_reads to genome h_sapiens_37_asm with Bowtie2
[sam_read1] missing header? Abort!
    [FAILED]
Error running bowtie:
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): bowtie2-align died with signal 6 (ABRT) (core dumped)

When I tried to view left_kept_reads.bam with samtools, it says that the file doesn't have an EOF marker

Any help would be much appreciated!