RSEM prepare reference error

Question

[billyconan6a]

Hi guys

I am now running a trial run on RSEM following the tutorial https://github.com/deweylab/RSEM, but when I type in this command

rsem-prepare-reference --gff3 p13.genomic.gff        --trusted-sources BestRefSeq,Curated\ Genomic        --star        primaryassembly.fna        ref/human_refseq

I got this

rsem-gff3-to-gtf p13.genomic.gff ref/human_refseq.gtf
Traceback (most recent call last):
  File "/usr/bin/rsem-gff3-to-gtf", line 249, in <module>
    fout = open(args.output_GTF_file, "w")
FileNotFoundError: [Errno 2] No such file or directory: 'ref/human_refseq.gtf'
"rsem-gff3-to-gtf p13.genomic.gff ref/human_refseq.gtf" failed! Plase check if you provide correct parameters/options for the pipeline!

My understanding on the command is that "ref/human_refseq" should be the output file name? but now it is saying no directory for "ref/human_refseq" so I really don't know what is going wrong.

Thank you very much.

Answer 1

[ATpoint]

You have to download that file and put it somewhere so the tool can access it.

Answer 2

[billyconan6a]

Thanks for the reply. I also encountered another problem, so after I type in this command

rsem-prepare-reference --gtf p13.gtf --trusted-sources "BestRefSeq, Curated Genomic" --star primaryassembly.fna output

The terminal returns

rsem-extract-reference-transcripts output 0 p13.gtf BestRefSeq,\ Curated\ Genomic 0 primaryassembly.fna
Parsed 200000 lines
Parsed 400000 lines
Parsed 600000 lines
Parsed 800000 lines
Parsed 1000000 lines
Parsed 1200000 lines
Parsed 1400000 lines
Parsed 1600000 lines
Parsed 1800000 lines
Parsed 2000000 lines
Parsed 2200000 lines
Parsed 2400000 lines
Parsed 2600000 lines
Parsed 2800000 lines
Parsed 3000000 lines
Parsed 3200000 lines
Parsed 3400000 lines
Parsed 3600000 lines
Parsed 3800000 lines
Parsing gtf File is done!
primaryassembly.fna is processed!
Warning: Cannot extract transcript NR_110202.1_1's sequence since the chromosome it locates, NW_003315913.1, is absent!
Warning: Cannot extract transcript NR_110203.1_1's sequence since the chromosome it locates, NW_003315913.1, is absent!
Warning: Cannot extract transcript NM_207365.4_1's sequence since the chromosome it locates, NW_003315913.1, is absent!
Warning: Cannot extract transcript NM_012138.4_1's sequence since the chromosome it locates, NT_187614.1, is absent!
Warning: Cannot extract transcript NM_003742.4_1's sequence since the chromosome it locates, NW_003315909.1, is absent!
Warning: Cannot extract transcript NM_004996.4_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NR_003569.1_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NR_023387.2_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NM_001079528.4_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NM_001171.6_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NM_001351800.1_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NR_147784.1_1's sequence since the chromosome it locates, NT_187607.1, is absent!
Warning: Cannot extract transcript NM_001025091.2_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NM_001090.3_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NM_001025091.2_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NM_001090.3_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NM_001025091.2_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NM_001090.3_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NM_001025091.2_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NM_001090.3_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NM_001025091.2_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NM_001090.3_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NM_001025091.2_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NM_001090.3_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NM_001177515.2_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NM_021160.3_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NR_033488.2_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NR_033489.2_1's sequence since the chromosome it locates, NT_113891.3, is absent!
Warning: Cannot extract transcript NM_001177515.2_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NM_021160.3_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NR_033488.2_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NR_033489.2_2's sequence since the chromosome it locates, NT_167245.2, is absent!
Warning: Cannot extract transcript NM_001177515.2_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NM_021160.3_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NR_033488.2_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NR_033489.2_3's sequence since the chromosome it locates, NT_167246.2, is absent!
Warning: Cannot extract transcript NM_001177515.2_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NM_021160.3_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NR_033488.2_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NR_033489.2_4's sequence since the chromosome it locates, NT_167247.2, is absent!
Warning: Cannot extract transcript NM_001177515.2_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NM_021160.3_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NR_033488.2_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NR_033489.2_5's sequence since the chromosome it locates, NT_167248.2, is absent!
Warning: Cannot extract transcript NM_001177515.2_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NM_021160.3_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NR_033488.2_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NR_033489.2_6's sequence since the chromosome it locates, NT_167249.2, is absent!
Warning: Cannot extract transcript NM_020469.3_1's sequence since the chromosome it locates, NW_009646201.1, is absent!
Warning: Cannot extract transcript NM_001092.5_1's sequence since the chromosome it locates, NT_187613.1, is absent!
Warning: 9752 transcripts are failed to extract because their chromosome sequences are absent.
84320 transcripts are extracted.
Extracting sequences is done!
Group File is generated!
Transcript Information File is generated!
Chromosome List File is generated!
Extracted Sequences File is generated!

rsem-preref output.transcripts.fa 1 output
Refs.makeRefs finished!
Refs.saveRefs finished!
output.idx.fa is generated!
output.n2g.idx.fa is generated!

STAR  --runThreadN 1  --runMode genomeGenerate  --genomeDir .  --genomeFastaFiles primaryassembly.fna  --sjdbGTFfile p13.gtf  --sjdbOverhang 100  --outFileNamePrefix output
Jun 05 11:52:36 ..... started STAR run
Jun 05 11:52:36 ... starting to generate Genome files
terminate called after throwing an instance of 'std::bad_alloc'
  what():  std::bad_alloc
"STAR  --runThreadN 1  --runMode genomeGenerate  --genomeDir .  --genomeFastaFiles primaryassembly.fna  --sjdbGTFfile p13.gtf  --sjdbOverhang 100  --outFileNamePrefix output" failed! Plase check if you provide correct parameters/options for the pipeline!

but when I run rsem-prepare-reference with bowtie with the following command, there was no such problem.

rsem-prepare-reference --gtf p13.gtf --trusted-sources "BestRefSeq, Curated Genomic" --bowtie primaryassembly.fna output

So why does my star just terminated itself?

Thanks.