blahah/transrate

Understand your transcriptome assembly

Rik

@blahah

the list of files to each argument should be comma separated, no spaces

you can also provide multiple assemblies and/or reference the same way

Rik

@blahah

@countloki thanks for reporting - we'll make a new release soon that will solve the salmon issues

Stuart Willis

@stuartwillis

@blahah and @cboursnell have you guys had any luck finding why salmon gives the "numeric overflow" error on some assemblies (issue #7 on the Transfuse github)? I have found it is reproducible running salmon on directly on the bam file.

Stuart Willis

@stuartwillis

at least one assembly also repeatably gives the following error, also from salmon: "terminate called after throwing an instance of 'std::bad_alloc'"

Rob Patro

@rob-p

@stuartwillis, any idea if that is still true with a newer salmon?

The version in transrate is very old at this point. Lots of improvements and bug fixes since.

@cboursnell @blahah, I'm booked this week, but perhaps we could work on integrating a newer salmon (and fixing other bugs) the following week, since some ppl seem to have recurring issues with 1.0.3

Stuart Willis

@stuartwillis

@rob-p, good question, I didn't try; I can check that if would justify upgrading transrate to a newer salmon

Rob Patro

@rob-p

Great. If you still see the error, please file a bug report for salmon so I can see if I can repro the issue upstream.

Stuart Willis

@stuartwillis

Indeed a more recent version of salmon (0.8.2) was successful on a bam/assembly that repeatedly resulted in a "numeric overflow" with 0.4.2.

However, I also found in the same case that deleting the transrate results (bam and all) and starting over circumvented the problem, while remaking the assembly (transabyss, in this case) circumvented this error in another case. Not sure what to make of that

It does speak to the prescience of @cboursnell to make transfuse able to pick up mid-way, though

Rik

@blahah

@rob-p definitely we should do that - I've been swamped for like 10 months, but this week I've finished up a project so I should have free cycles for it

wow we are super out of sync with salmon

@rob-p how close are we to being able to skip SNAP altogether? in-memory streaming alignment -> assignment -> assessment is the dream we talked about a year ago

Rob Patro

@rob-p

We are ~90% of the way done I'd say. I have 2 students working on this (which also includes some algorithmic advances to rapmap).

Their stuff adds the ability to "selectively" align quasi-mappings. Given that, it'll be a few days of work on the salmon end to enable the posterior sampling with the new code.

Rik

@blahah

@rob-p sweet - that will be such a great update

Stuart Willis

@stuartwillis

if you guys are tweaking the implementation of salmon into transrate, would it be worth it to add a specification for strand specific data? Not sure how salmon's behavior changes w or w/o on stranded data

jdeyar

@jdeyar

Hello @blahah,

Referring to other posts regarding an issue with 1.0.3, I am having a similar SNAP error while running transrate. My error code is noted below.

Welcome to SNAP version 1.0dev.96.

mmap failed.
MemMapDataReader: fail to map /pylon2/bi4s8fp/jsdsmail/Transrate/Trim_TS6_T24.left.pe.qc.fq at 0,3765724304
SNAP exited with exit code 1 from line 2283 of file SNAPLib/DataReader.cpp

Any help/advice on this error would be much appreciated. I got the same error when trying to run it on the previous version of transrate as well.

Thanks!

Rik

@blahah

@jdeyar hmm sorry about that, I'm going to try to work through the transrate issues next week (I'm no longer paid to work on it, and have been swamped with other stuff for a while)

@jdeyar hopefully this is one we can get dealt with quickly by just updating SNAP and Salmon and cleaning up a few packaging issues

jdeyar

@jdeyar

@blahah thank you for your prompt response. Hopefully it is as easy as an update for SNAP and other linked software. Please comment in this forum whenever it is updated! Much appreciated!

XinpengQi

@XinpengQi

@blahah any updates so for? I am running Transrate v1.0.3. The algorithms for contig/assembly score are so attractive! Eager to try them out in my data! SNAP seems fine, but Salmon gives warning "[jointLog] [warning] [Sampler.hpp]: Failed to sample an alignment for this read; this shouldn't happen
currentMass = -nan, r = 0.694954"

snap.log:

Loading index from directory... 2s. 384271831 bases, seed size 23
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns Extra Alignments %Pairs Reads/s Time in Aligner (s)
693,340,846 280,486,471 (40.45%) 330,208,110 (47.63%) 78,660,098 (11.35%) 3,986,167 (0.57%) 620,752,208 78.36% 124,387 5,574
Welcome to SNAP version 1.0dev.96.

salmon.log

[2017-05-17 13:40:55.996] [fileLog] [info] quantification processed 354000000 fragments so far
[2017-05-17 13:41:01.787] [jointLog] [info]
Completed first pass through the alignment file.
Total # of mapped reads : 354762625
of uniquely mapped reads : 144015998
ambiguously mapped reads : 210746627
[2017-05-17 13:41:01.761] [fileLog] [info] quantification processed 354762625 fragments so far
[2017-05-17 13:41:33.214] [jointLog] [info] Computed 9628374 rich equivalence classes for further processing
[2017-05-17 13:41:33.214] [jointLog] [info] Counted 354762625 total reads in the equivalence classes
[2017-05-17 13:41:33.227] [jointLog] [info] starting optimizer
[2017-05-17 13:41:39.341] [jointLog] [info] Marked 0 weighted equivalence classes as degenerate
[2017-05-17 13:41:39.618] [jointLog] [info] iteration = 0 | max rel diff. = 800.763
[2017-05-17 13:41:53.311] [jointLog] [info] iteration 50, recomputing effective lengths
[2017-05-17 13:42:09.716] [jointLog] [info] iteration = 100 | max rel diff. = 0.358082
[2017-05-17 13:42:40.040] [jointLog] [info] iteration = 200 | max rel diff. = 0.305365
[2017-05-17 13:43:10.754] [jointLog] [info] iteration = 300 | max rel diff. = 0.0754343
[2017-05-17 13:43:41.135] [jointLog] [info] iteration = 400 | max rel diff. = 0.0490107
[2017-05-17 13:44:09.308] [jointLog] [info] iteration 500, recomputing effective lengths
[2017-05-17 13:44:10.037] [jointLog] [info] iteration = 500 | max rel diff. = 0.0219555
[2017-05-17 13:44:39.741] [jointLog] [info] iteration = 600 | max rel diff. = 0.127602
[2017-05-17 13:45:09.829] [jointLog] [info] iteration = 699 | max rel diff. = 0.0077808
[2017-05-17 13:45:09.864] [jointLog] [info] finished optimizer
[2017-05-17 13:45:10.734] [jointLog] [info] Sampling alignments; outputting results to ./postSample.bam
[2017-05-17 13:45:11.509] [jointLog] [warning] [Sampler.hpp]: Failed to sample an alignment for this read; this shouldn't happen
currentMass = -nan, r = 0.694954

Rik

@blahah

cc @rob-p \o any ideas?

Rob Patro

@rob-p

Yes (cc @blahah), this is a bug that can occur in VB inference that was fixed in a more recent version of Salmon. I fear the only immediate solution here may be to generate the relevant file manually with a more recent Salmon release --- though we should probably upgrade the one included in Transrate soon

btw, the preprint that describes the relevant algorithm for having salmon do alignment internally went up yesterday --- http://biorxiv.org/content/early/2017/05/17/138800

I think that we can do a patch release before that, but this (rather big new feature) could land soon.

Rik

@blahah

so @rob-p if I cut a new transrate with updated salmon it will fix this?

Rob Patro

@rob-p

This should be the case, yes --- however, before you do that, we should really test the new pipeline on one or two data sets first. A lot has been updated, but the module to sample alignments hasn't been touched in a while.

Rik

@blahah

XinpengQi

@XinpengQi

Thanks! @blahah @rob-p If any testing needed from users, I would like to do it.

JakeGoodall

@JakeGoodall

Hi Transrate community. I'm hoping someone might be able to provide some advice. Similar to @Victaphanta and @sarjopp earlier in the thread I'm having issues installing all dependencies (namely Salmon_v0.4.0) using the "transrate --install-deps=all" command. As suggested further up I've tried both deleting and renaming local/bin/librt.so.1, both of which did not help. Did this issue ever get resolved? Or at the very least is there an existing/ reliable method to get salmon0.4.0 working?

Matt MacManes

@macmanes

can I hope that when TransRate is updated, @cboursnell can update TransFuse as well? Both of these are really important to my workflow, and are unreliable.. Transfuse is still using Salmon 0.4.2, giving me intractable errors

[ERROR] 2017-05-23 13:47:30 : Version Info: Could not resolve upgrade information in the alotted time.
Check for upgrades manually at https://combine-lab.github.io/salmon
# salmon (alignment-based) v0.4.2
# [ program ] => salmon
# [ command ] => quant
# [ libType ] => { IU }
# [ alignments ] => { /mnt/lustre/macmaneslab/macmanes/orp/assemblies/transrate_1.transcripts75/1.skewer-trimmed-pair1.fastq.1.skewer-trimmed-pair2.fastq.1.transcripts75.bam }
# [ targets ] => { /mnt/lustre/macmaneslab/macmanes/orp/assemblies/1.spades_k75/1.transcripts75.fasta }
# [ threads ] => { 24 }
# [ sampleOut ] => { }
# [ sampleUnaligned ] => { }
# [ output ] => { . }
# [ useVBOpt ] => { }
# [ useErrorModel ] => { }
Library format { type:paired end, relative orientation:inward, strandedness:unstranded }
Logs will be written to ./logs
numQuantThreads = 18
parseThreads = 6
Checking that provided alignment files have consistent headers . . . done
Populating targets from aln = "/mnt/lustre/macmaneslab/macmanes/orp/assemblies/transrate_1.transcripts75/1.skewer-trimmed-pair1.fastq.1.skewer-trimmed-pair2.fastq.1.transcripts75.bam", fasta = "/mnt/lustre/macmaneslab/macmanes/orp/assemblies/1.spades_k75/1.tra
nscripts75.fasta" . . .done




processed 947385 reads in current round

/mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/ruby/2.2.0/gems/transrate-1.0.1/lib/transrate/salmon.rb:27:in `run': Salmon failed (Transrate::SalmonError)
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/ruby/2.2.0/gems/transrate-1.0.1/lib/transrate/read_metrics.rb:120:in `assign_and_quantify'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/ruby/2.2.0/gems/transrate-1.0.1/lib/transrate/read_metrics.rb:63:in `run'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/ruby/2.2.0/gems/transrate-1.0.1/lib/transrate/transrater.rb:86:in `read_metrics'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:218:in `block (2 levels) in transrate'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:214:in `chdir'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:214:in `block in transrate'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:210:in `each'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:210:in `each_with_index'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/libexec/lib/lib/transfuse/transfuse.rb:210:in `transrate'
    from /mnt/lustre/software/linuxbrew/colsa/Cellar/transfuse/86.64/bin/../libexec/lib/bin/transfuse:66:in `<main>'
make: *** [transfuse] Error 1

the Salmon log simply says:

[2017-05-23 13:47:28.703] [fileLog] [info] quantification processed 0 fragments so far

[2017-05-23 13:47:28.948] [fileLog] [info] quantification processed 947385 fragments so far

@rob-p @blahah

Rob Patro

@rob-p

wow --- that looks like a hard fail. Presumably a segfault, but I can't really assess from this output.

Matt MacManes

@macmanes

ya, and it’s reproducible… and 100% not worth chasing since it’s a version from 9034 years ago..

Rob Patro

@rob-p

l0l --- true. But presumably we could verify that if we run with 0.8.2 (same index and alignments) everything works?

Matt MacManes

@macmanes

Just did that.. can confirm that everything runs perfect in 0.8.2. :)

Rob Patro

@rob-p

great :) --- we'll get that fixed as soon as Richard and I can sync up. I think getting this issue resolved asap is important. The awesome selective-alignment stuff should probably wait until the subsequent release (after it's had sufficient testing within Salmon itself) ;P.

tawares07

@tawares07

Hi guys! I'm new in Linux and Transcriptome assembly. I downloaded Transrate but I really don't know how to install it. I did a "make" command but nothing happened. So, how can I install and run Transrate? (I know it's a silly question but as I told you, I'm new in Transcriptome Assembly. I hope you understand

Matt MacManes

@macmanes

There are some instructions, here, that might help: http://oyster-river-protocol.readthedocs.io/en/master/aws_setup.html

Rik

@blahah

ok so a straight salmon update to 0.8.2 should solve some things

easy to do

will try to push that out this weekend

Ilya Shlyakhter

@notestaff

Can you add a bioconda recipe for installing transrate? I've done some work on it ( https://github.com/bioconda/bioconda-recipes/tree/is-add-transrate/recipes/transrate ) but it's not working yet. The transrate gem on rubygems.org is 1.0.1 but the current version of transrate is 1.0.3 . The dependence on snap-aligner is on 1.0dev.96 , but would 1.0dev.97 work? bioconda has a build for 1.0dev.97 . Would blast+ 2.2.31 work? bioconda has a build for that, but not for 2.2.29 . In bioconda the guideline is to have a recipe for building things from source, so just fetching binaries would not work.

Austin Patton

@austinhpatton

Hi there - I've been using both Transrate and Transfuse in my assembly pipeline. In brief, I've been using three different assemblers choosing different settings within each. I then ranked each resultant assembly with Transrate and merged both within an across assemblers using Transfuse. Out of interest, I re-ranked each merged assembly using the recent version of Transrate, and what I found was that their scores did not correspond to those reported by Transfuse - they were much lower, and lower than any of the original assemblies.

I'm wondering why this might be? How does Transfuse's merging procedure differ from that of the merging implemented within Transrate? Is one preferred over the other?

Also, I haven't been able to find any documentation describing in detail what exactly Transfuse is doing. Is this available somewhere?

Thanks!

pchelych

@pchelych_twitter

Anybody home?

preeti333

@preeti333

Does this software works for paired end data only?

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snap.log:

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