# Trinity align reads and estimate abundance Fatal Error **URL:** \ **Category:** usegalaxy.org support\ **Tags:** assembly, troubleshooting, salmon\ **Created:** [June 22, 2020, 8:37pm UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961 "2020-06-22T20:37:40Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![h.mckay](https://avatars.discourse-cdn.com/v4/letter/h/46a35a/32.png) [@h.mckay](https://help.galaxyproject.org/u/h.mckay)\ **Post date:** [June 22, 2020, 8:37pm UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/1 "2020-06-22T20:37:40Z")
Hi, I’ve recently encountered an error when running the ‘align reads and estimate abundance’ tool in the Trinity de novo RNA-seq analysis pipeline. The output is as follows: Fatal error: Exit code 2 () CMD: touch /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie.started CMD: bowtie-build /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie CMD: touch /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.RSEM.rsem.prepped.started CMD: rsem-prepare-reference --transcript-to-gene-map /galaxy-repl/main/jobdir/028/998/28998407/working/gene\_to\_trans.map /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.RSEM $VAR1 = [ { ‘right’ =\> ‘/galaxy-repl/main/jobdir/028/998/28998407/working/paired\_right.fq’, ‘left’ =\> ‘/galaxy-repl/main/jobdir/028/998/28998407/working/paired\_left.fq’, ‘output\_dir’ =\> ‘output’ } ]; CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 6 /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie -1 /galaxy-repl/main/jobdir/028/998/28998407/working/paired\_left.fq -2 /galaxy-repl/main/jobdir/028/998/28998407/working/paired\_right.fq | samtools view -@ 6 -F 4 -S -b | samtools sort -@ 6 -n -o bowtie.bam Warning: Skipping pair SRR5112684.25647332/1 HWI-D00525:102:C4NM2ANXX:2:2302:6220:36668 length=125/1 because a mate is less than 4 characters long … 4NM2ANXX:2:2106:14360:26107 length=125/1 because a mate is less than 4 characters long # reads processed: 917268 # reads with at least one reported alignment: 650697 (70.94%) # reads that failed to align: 266571 (29.06%) Reported 1024386 paired-end alignments [bam\_sort\_core] merging from 0 files and 6 in-memory blocks… CMD: touch bowtie.bam.ok CMD: convert-sam-for-rsem bowtie.bam bowtie.bam.for\_rsem Number of first and second mates in read SRR5112684.97/1’s full alignments (both mates are aligned) are not matched! Error, cmd: convert-sam-for-rsem bowtie.bam bowtie.bam.for\_rsem died with ret: 65280 at /cvmfs/main.galaxyproject.org/deps/\_conda/envs/mulled-v1-3213810583b3c414a873752c2610a95351f8665124407340352879200d8f7bbf/bin/align\_and\_estimate\_abundance.pl line 729. I’ve found an answer to a similar question from the galaxy biostar page ([Red error from RSEM Trinity -- Solution: input matched paired end reads](https://biostar.galaxyproject.org/p/28961/)) which suggested there were different numbers of reads between the pairs, but when I checked with FastQ Interlacer, there were no single reads (all reads were matched between F & R). If anyone has any suggestions regarding this issue, that would be greatly appreciated. Thanks ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [June 22, 2020, 9:18pm UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/2 "2020-06-22T21:18:15Z")
Hello @h.mckay Did you run any QA on these data? `Trimmomatic` can eliminate the very short reads (reported as warnings by `Trinity`) and will result in 4 outputs – 2 datasets for forward + reverse that are still paired after QA and 2 for those that are not. You may choose to do more with `Trimmomatic` (remove adaptor, etc) – run `FastQC` on your data to review original quality and decide. It is common to run `FastQC` both before and after `Trimmomatic`, to compare results/success of the QA filtering applied. Once done, input the “still paired reads” to `Trinity` and see if that resolves the error. Thanks! ---
**Author:** ![h.mckay](https://avatars.discourse-cdn.com/v4/letter/h/46a35a/32.png) [@h.mckay](https://help.galaxyproject.org/u/h.mckay)\ **Post date:** [June 22, 2020, 9:56pm UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/3 "2020-06-22T21:56:41Z")
Hi @jennaj I did run FastQC both pre- and post-Trimmomatic on my data, which eliminated failures, but maintained a few warnings: per sequence GC content, sequence length distribution (a very small percentage were a different length than the rest, graph was a tight peak at ~115bp), and sequence duplication levels (28%). I can’t seem to find if there are specific quality requirements for running Trinity, but there were no warnings in the trinity log (assembly ran successfully). If you think I should make my quality cutoffs more stringent, please let me know. Thanks! ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [June 23, 2020, 1:40am UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/4 "2020-06-23T01:40:01Z")
Filtering read by quality (20 == 99% base-calling accuracy) and length (remove very short sequences) will make the assembly cleaner and less likely to run into resource problems. This means that the QA needs to be done on the reads used to generate the `Trinity` assembly and the reads mapped back against that assembly result. > [@h.mckay](#): > > Number of first and second mates in read SRR5112684.97/1’s full alignments (both mates are aligned) are not matched! This part of the error message seemed suspicious given that your reads are paired up, so I web searched and found that others ran into this problem when using the option `RSEM with Bowtie`. [Trinity transcript\_abundance.pl error using RSEM · Issue #305 · trinityrnaseq/trinityrnaseq · GitHub](https://github.com/trinityrnaseq/trinityrnaseq/issues/305) Some resolved it by using `RSem with Bowtie2` instead. Maybe try a rerun using the `Bowtie2` option? You can toggle between `Bowtie` and `Bowtie2` in the Galaxy wrapped version of the tool. A highly fragmented Trinity assembly could also lead to this problem (or that is my guess – if the assembled contigs do not represent full transcripts, then reads could map to different contigs and then “not match”) – but it is definitely worth a try to see if `Bowtie2` works first. You may also want to review the assembly itself, and potentially filter it by length. Any contig that is shorter than your reads will not capture hits, but contigs that are only long enough to capture one end of the read pair can lead to a multi-mapping issue. How long should a valid contig be? It depends on the species/genome – but any “contig” that is really short (under ~100 bases, or the length of the original reads) could represent data that wasn’t incorporated into a full-length transcript. That could be related to the original read quality, untrimmed artifact, or potentially the depth or coverage of sequencing was low. Even upstream issues with how the library was prepared could be a factor. Meaning, it is possible that full length alternatively spliced contigs (transcripts) were not generated. This could lead to mapping problems … _especially_ if the same reads were used to generate the assembly that were later mapped back to it (a “poor” quality read would map back against itself). The goal is to create a high-quality assembly that does not include partial/duplicated content. Please review your original assembly reads, clean those up and reassemble as needed, then try `Bowtie2` with reads that also were quality filtered, to eliminate read quality and technical factors, and we can follow up from there regarding assembly content as needed. Note: `Trinity` is currently problematic at [usegalaxy.org](http://usegalaxy.org), but we expect to have that resolved as a priority fix. Related Q&A about that (`Trinity` runs on the same cluster as these other tools): [SPADES - Remote job server indicated a problem running or monitoring this job. - #10 by jennaj](https://help.galaxyproject.org/t/spades-remote-job-server-indicated-a-problem-running-or-monitoring-this-job/3934/10) Apologies for the confusion, but we can sort out the different issues, and doing more QA first is probably needed anyway. Thanks! ---
**Author:** ![h.mckay](https://avatars.discourse-cdn.com/v4/letter/h/46a35a/32.png) [@h.mckay](https://help.galaxyproject.org/u/h.mckay)\ **Post date:** [June 23, 2020, 2:12am UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/5 "2020-06-23T02:12:38Z")
Hi @jennaj Thanks for the info, but unfortunately, switching Bowtie to Bowtie2 resulted in the same error. I was also confused about the ‘not matched!’ error, since I confirmed all my reads were matched with fastq interlacer. How would I determine if my Trinity assembly is highly fragmented, and if it is, is there a way to fix it? Thanks ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [June 23, 2020, 2:33am UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/6 "2020-06-23T02:33:59Z")
If your reads were not quality trimmed before assembly, then you should do that first. Resources about the `Trinity` pipeline, include assembly statistics tools: > [@What to do with trinity output files](https://help.galaxyproject.org/t/what-to-do-with-trinity-output-files/2497/2): > > Welcome @Priya_GNDU! Tool: Build expression matrix for a de novo assembly of RNA-Seq data by Trinity Resources for generating/working with Trinity assemblies: Author’s Wiki, includes links to tutorials/resources: [Home · trinityrnaseq/trinityrnaseq Wiki · GitHub](https://github.com/trinityrnaseq/trinityrnaseq/wiki)[Accessing Trinity on Publicly Available Compute Resources · trinityrnaseq/trinityrnaseq Wiki · GitHub](https://github.com/trinityrnaseq/trinityrnaseq/wiki/Accessing-Trinity-on-Publicly-Available-Compute-Resources)[Home · trinityrnaseq/GalaxyTrinityProtocol Wiki · GitHub](https://github.com/trinityrnaseq/GalaxyTrinityProtocol/wiki) The Trinity command-line tools are wrapped for Galaxy. Help is on eac… > [@What are the RNA-seq data processing steps in Galaxy according to de novo approach?](https://help.galaxyproject.org/t/what-are-the-rna-seq-data-processing-steps-in-galaxy-according-to-de-novo-approach/3943/2): > > Hi @f_kurt Please see this prior Q&A, it covers most of what you are asking about. It is worth reviewing first: [What to do with trinity output files](https://help.galaxyproject.org/t/what-to-do-with-trinity-output-files/2497) For tools that appear to be missing, it could be that the Galaxy server you are working at does not have them installed. Or, the tool panel search is not finding them for some reason (search term typo?). You can also directly check. Trimmomatic is often under the tool group “FASTQ Quality Control”. And yes, performing QA is probably a good idea… That said, I still think this could be a server-side issue. These tools were designed to work with data that isn’t “perfect”. You could also try alternative options than `RSEM` – `Salmon` or `Kalisto` as others needed to do (see the ticket that the `Trinity` authors wrote back on [Trinity transcript\_abundance.pl error using RSEM · Issue #305 · trinityrnaseq/trinityrnaseq · GitHub](https://github.com/trinityrnaseq/trinityrnaseq/issues/305)). But if this is an actual cluster issue, those wouldn’t work either. In testing today, many tools that run on the same cluster as this one all failed. Our administrator is working on it. ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [December 21, 2023, 12:09am UTC](https://help.galaxyproject.org/t/trinity-align-reads-and-estimate-abundance-fatal-error/3961/7 "2023-12-21T00:09:23Z")