Estimating Transcript Abundance with Salmon

[Mauricio Losilla]

Hi,

I am trying to use Trinity's script align_and_estimate_abundance.pl to quantitate transcript abundance with Salmon. This is my command and versions of the programs:

module load GNU/4.8.2

module load salmon/0.6.0

module load trinity/2.2.0

$TRINITY_HOME/util/align_and_estimate_abundance.pl --transcripts ../Trinity-GG.fasta --seqType fq --left ${PBS_O_WORKDIR}/../../NormTrinityOutput/insilico_read_normalization/left.norm.fq --right ${PBS_O_WORKDIR}/../../NormTrinityOutput/insilico_read_normalization/right.norm.fq --est_method salmon --trinity_mode --output_dir ${PBS_O_WORKDIR} --SS_lib_type RF --prep_reference

This command runs fine for a while, but then I get this:

Greater than 5% of the alignments (but not, necessarily reads) disagreed with the provided library type; check the file: /......../GGTrinityOutput/Salmon/libFormatCounts.txt for details

Error, cmd: salmon quant -i /...../GGTrinityOutput/Salmon/../Trinity-GG.fasta.salmon_quasi.idx -l ISR -1 /...../GGTrinityOutput/Salmon/../../NormTrinityOutput/insilico_read_normalization/left.norm.fq -2 /........../GGTrinityOutput/Salmon/../../NormTrinityOutput/insilico_read_normalization/right.norm.fq -o /......./GGTrinityOutput/Salmon  -p 4 died with ret: 139 at /opt/software/trinity/2.2.0--GCC-4.8.2/util/align_and_estimate_abundance.pl line 743.

The highlights of the file libFormatCounts.txt are (I deleted a bunch of lines with zeros):

Expected format: Library format { type:paired end, relative orientation:inward, strandedness:(antisense, sense) }

# of consistent alignments: 96522716

# of inconsistent alignments: 16585238

========

---- counts for each format type ---

.

.

.

Library format { type:paired end, relative orientation:outward, strandedness:(sense, antisense) } : 1130

Library format { type:single end, relative orientation:inward, strandedness:(sense, antisense) } : 0

Library format { type:paired end, relative orientation:inward, strandedness:(sense, antisense) } : 2011808

.

.

.

Library format { type:paired end, relative orientation:outward, strandedness:(antisense, sense) } : 49155

Library format { type:single end, relative orientation:inward, strandedness:(antisense, sense) } : 0

Library format { type:paired end, relative orientation:inward, strandedness:(antisense, sense) } : 96522716

.

.

.

Library format { type:paired end, relative orientation:matching, strandedness:sense } : 2381

.

.

.

Library format { type:single end, relative orientation:none, strandedness:sense } : 6939095

Library format { type:paired end, relative orientation:none, strandedness:sense } : 0

Library format { type:single end, relative orientation:matching, strandedness:antisense } : 0

Library format { type:paired end, relative orientation:matching, strandedness:antisense } : 46450

.

.

.

Library format { type:single end, relative orientation:none, strandedness:antisense } : 7535219

.

.

.

I contacted Trinity's developers but they don't know how to get around this.

Any idea of what is wrong?

Thanks

Mau