Trinity de novo transcriptome assembly: samtools view failed to add PG line to the header

[Fairo]

Dear all,

I hope this message find you well.

I have assembled the transcriptome a plant species using Trinity.

Here is the command:
Trinity --samples_file ./sample_file.txt --seqType fq --max_memory 80G --CPU 30 --output ./trinity_outdir

I tried to get transcript abundance using the following command:
/home/programs/build/trinityrnaseq/util/align_and_estimate_abundance.pl --transcripts ./Trinity.fasta --seqType fq --samples_file ./sample_file.txt --thread_count 6 --gene_trans_map ./trinity_outdir.Trinity.fasta.gene_trans_map --est_method RSEM --aln_method bowtie2 --prep_reference --coordsort_bam --output_dir /rsem_outdir/

And I keep getting the following error:
[E::sam_hrecs_update_hashes] Duplicate entry "TRINITY...." in sam header
samtools view: failed to add PG line to the header


I will appreciate your suggestions.

[Brian Haas]

Hi,

My suggestion would be to just run salmon or kallisto and see how that goes. It should be much faster and use less resources.

best,

~b

[Fairo]

Thank you for your reply, B.

I have tried but it failed. Salmon requires the genome of the organism in both modes. However, I do not have the genome yet. Will it be wise if I remove the duplicated from the trintiy. fasta? I have a single organism with three different tissues.

[Brian Haas]

Hi,

Salmon shouldn't need the genome - just needs the Trinity fasta file and the reads you want to use for quantification.  We support it exactly the same as the other quant methods:

https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Transcript-Quantification

best,

~b

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

 

[DAYOU Olivier]

Thank you, Brian.

I have tried the align_and_estimate_abundance.pl with --est_method salmon.

But it fails, here is the error message.

[Step 1 of 4] : counting k-mers
[2022-08-12 12:58:33.007] [puff::index::jointLog] [error] In FixFasta, two references with the same name but different sequences: TRINITY_DN11452_c0_g1_i3. We require that all input records have a unique name up to the first whitespace (or user-provided separator) character.
Error, cmd: salmon index -t /home/organism ./Trinity.fasta --keepDuplicates -i /home/organism/./Trinity.fasta.salmon.idx -k 31 -p 6 died with ret: 256 at /home/programs/build/trinityrnaseq/util/align_and_estimate_abundance.pl line 729.

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Olivier Dayou| Skype:  Olivier DAYOU                           

[DAYOU Olivier]

There was also a warning:

[warning] The salmon index is being built without any decoy sequences.  It is recommended that decoy sequence (either computed auxiliary decoy sequence or the genome of the organism) be provided during indexing.

[Brian Haas]

It's quite strange that the identifier TRINITY_DN11452_c0_g1_i3 is apparently showing up multiple times.

Are you running salmon on the results from a single Trinity assembly, or did you perhaps concatenate several together?

[DAYOU Olivier]

I running salmon on a single trinity assembly generated directly by trinity from three different tissues of the same organism. It is not a concstenated assembly.

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[Brian Haas]

Interesting.

If you grep "TRINITY_DN11452_c0_g1_i3" from the Trinity.fasta file, are you finding multiple records for it?

[DAYOU Olivier]

Yes, grep TRINITY_DN11452_c0_g1_i3 Trinity.fasta outputs two of the same patterns/ids

[Brian Haas]

That's very peculiar.  Not supposed to happen.

Try rerunning your original Trinity command. I expect it should just remake the final Trinity.fasta file.  Then, let's see if still contains duplicate entries for this one.