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. 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

.

.

.

Any idea of what is wrong?

Thanks

Mau

[Brian Haas]

Hi Mau,

Try dropping the --SS_lib_type RF  and let's see if it works.   

~b

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.
To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.
To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

[Mauricio Losilla]

Hi Brian,

Thanks for your assistance. I tried it but didn't work. This is the error message:

Detected a strand bias > 1% in an unstranded protocol check the file: /......./GGTrinityOutput/Salmon/libFormatCounts.txt for details

.

.

.

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

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:unstranded }

# of consistent alignments: 98534524

# of inconsistent alignments: 14573430

strand bias = 0.979583 (0.5 is unbiased)

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

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

========

---- 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:single end, relative orientation:none, strandedness:(antisense, sense) } : 0

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

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

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:outward, strandedness:antisense } : 0

Library format { type:paired end, relative orientation:outward, strandedness:antisense } : 0

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

Library format { type:paired end, relative orientation:inward, strandedness:antisense } : 0

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

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Brian Haas]

That's too bad.  I was hoping that Salmon would be a good fit for use w/ de novo transcriptome data, but it might need some configuring to enable it.   Can you check with the Salmon developers regarding this (or I certainly can)?

In the meantime, eXpress, RSEM, and kallisto should be compatible with what you have.

best,

~b

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--

You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Mauricio Losilla]

Maybe I should have mentioned that this is a genome guided assembly, if that matters.

I could certainly post on the sailfish google group if you think that would help

Mau

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Brian Haas]

Shouldn't matter.  Let's see what they say

Thx

-Brian

(by iPhone)

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

[Mauricio Losilla]

I posted on their group 6 days ago, but got no response.

https://groups.google.com/forum/#!topic/sailfish-users/HfCbWFwknUU

I used Kallisto instead. 

Thanks

[Brian Haas]

Good to know.   I'm glad to hear that kallisto worked well for you.

~b

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-u...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Rob Patro]

Hi all,

  I'm sorry I missed your post on our group, Mauricio.  I try to respond quickly, but sometimes I miss certain posts.  Anyway, I just wanted to note that the message you mentioned above with salmon is not  an error — it's simply to inform you about the fraction of reads that can map in the given orientation.  Since strand-specific libraries are often not perfectly precise, it's often the case that while most reads will map in accordance with the library types not all will.  The libFormatCounts.txt file you specify summarize these mapping types for you.  That being said, the default mode in previous versions of salmon was a bit too conservative in that it simply considers and reports library compatibility for all possible mappings.  What you really want to know, and what we now report, is the number of fragments that have at least one mapping compatible with the specified library type, and the number of fragments that are incompatible (i.e. have no compatible mapping).  This is a more liberal criterion.  However, even so, when you get this warning message it's just to inform you that the stranded library's specificity may be lower than ideal, but it's not necessarily a problem.  Relatedly, (you may be interested to know this Brian), as of v0.7.0 we support automatic library type detection with the `-l A` option, so that Salmon will automatically infer the most likely library type from the mappings of the reads.

--Rob

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Brian Haas]

Thanks, Rob!   It looks like the earlier version exited with non-zero in this case.  Does the latest version exit (0) when this issue occurs?  We use the exit value to define success/failure.

much appreciated,

~brian

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsubscribe...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsubscribe...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsubscribe...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsubscribe...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.
To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrnaseq-users@googlegroups.com.

Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

[Rob Patro]

Hi Brian,

  Actually, the return code, even when there is a strand bias should have been 0.  If you were seeing a non-zero return code, my guess is that Mauricio was running into the issue described in the "Note" here.  This was resolved a while ago with a patch to 0.6.0 and is thus also fixed in subsequent versions.  Basically, there was an issue with the JEMalloc allocator where, on certain system configurations, a double-free would occur during program shutdown.  All of the output was OK, but it obviously affected the program return code.  Anyway, this issue has been resolved for a while.  All normal executions of Salmon should return 0.

Best,

Rob

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.
To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.
To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.
To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.

To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.
To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

--
You received this message because you are subscribed to the Google Groups "trinityrnaseq-users" group.
To unsubscribe from this group and stop receiving emails from it, send an email to trinityrnaseq-users+unsub...@googlegroups.com.

To post to this group, send email to trinityrn...@googlegroups.com.
Visit this group at https://groups.google.com/group/trinityrnaseq-users.
For more options, visit https://groups.google.com/d/optout.