What is the requirement for bam input of rsem-eval-calculate-score?

[wangyugui]

What is the requirement for bam input of rsem-eval-calculate-score?

In the following samples, the bam is generate by Trinity/${TRINITY_HOME}/util/align_and_estimate_abundance.pl --est_method RSEM --aln_method bowtie2.

and this bam file is sorted.

+ rsem-eval-estimate-transcript-length-distribution /biowrk/juglans.hybrid.mRNA/trinity/Transcriptome.fa human.txt
+ '[' 0 '!=' 0 ']'
+ rsem-eval-calculate-score -p 48 --transcript-length-parameters human.txt --bam --paired-end /biowrk/juglans.hybrid.mRNA/trinity.est/A0/A0-1/sort.bam /biowrk/juglans.hybrid.mRNA/trinity/Transcriptome.fa A0-1 180
rsem-synthesis-reference-transcripts A0-1.temp/A0-1 0 0 0 /biowrk/juglans.hybrid.mRNA/trinity/Transcriptome.fa
Transcript Information File is generated!
Group File is generated!
Extracted Sequences File is generated!

rsem-preref A0-1.temp/A0-1.transcripts.fa 1 A0-1.temp/A0-1
Refs.makeRefs finished!
Refs.saveRefs finished!
A0-1.temp/A0-1.idx.fa is generated!
A0-1.temp/A0-1.n2g.idx.fa is generated!

rsem-parse-alignments A0-1.temp/A0-1 A0-1.temp/A0-1 A0-1.stat/A0-1 b /biowrk/juglans.hybrid.mRNA/trinity.est/A0/A0-1/sort.bam -t 3 -tag XM
The SAM/BAM file declares more reference sequences (1197667) than RSEM knows (134092)!
"rsem-parse-alignments A0-1.temp/A0-1 A0-1.temp/A0-1 A0-1.stat/A0-1 b /biowrk/juglans.hybrid.mRNA/trinity.est/A0/A0-1/sort.bam -t 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
+ '[' 255 '!=' 0 ']'

[kubu4]

I know this issue is a few years old, but I'll post here to add support in hopes of getting a response, as I'm encountering a similar problem with a different error that I can't interpret. I'm using a sorted BAM file (generated with Bowtie2, using the alignment params recommended by DETONATE):

rsem-synthesis-reference-transcripts cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta 0 0 0 /gscratch/srlab/sam/data/C_bairdi/transcriptomes/cbai_transcriptome_v3.1.fasta
Transcript Information File is generated!
Group File is generated!
Extracted Sequences File is generated!

rsem-preref cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta.transcripts.fa 1 cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta
Refs.makeRefs finished!
Refs.saveRefs finished!
cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta.idx.fa is generated!
cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta.n2g.idx.fa is generated!

rsem-parse-alignments cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta cbai_transcriptome_v3.1.fasta.stat/cbai_transcriptome_v3.1.fasta b /gscratch/scrubbed/samwhite/outputs/20201224_cbai_bowtie2_transcriptomes_alignments/cbai_transcriptome_v3.1.fasta.sorted.bam -t 3 -tag XM
rsem-parse-alignments: parseIt.cpp:92: void parseIt(SamParser*) [with ReadType = PairedEndReadQ; HitType = PairedEndHit]: Assertion `val == 0 || val == 1 || val == 5' failed.
"rsem-parse-alignments cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta cbai_transcriptome_v3.1.fasta.temp/cbai_transcriptome_v3.1.fasta cbai_transcriptome_v3.1.fasta.stat/cbai_transcriptome_v3.1.fasta b /gscratch/scrubbed/samwhite/outputs/20201224_cbai_bowtie2_transcriptomes_alignments/cbai_transcriptome_v3.1.fasta.sorted.bam -t 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

Here's what the head of the BAM file looks like:

[samwhite@n2233 20201224_cbai_bowtie2_transcriptomes_alignments]$ /gscratch/srlab/programs/samtools-1.10/samtools view cbai_transcriptome_v3.1.fasta.sorted.bam | head
A00147:108:HLLJFDMXX:1:1369:3893:6637	163	TRINITY_DN5604_c0_g2_i1	1	42	101M	=	81	181	GAAAGAAAAACCGACAGGAGGAATTTCTTTGTTACCAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAA	:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFF	AS:i:-2	XN:i:0	XM:i:2	XO:i:0	XG:i:0	NM:i:2	MD:Z:2G31A66	YS:i:0	YT:Z:CP
A00147:121:HLLVMDMXX:1:2159:5674:25786	163	TRINITY_DN5604_c0_g2_i1	4	42	101M	=	72	169	AGAAAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTA	FFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP
A00147:108:HLLJFDMXX:2:1420:13702:14920	163	TRINITY_DN5604_c0_g2_i1	5	42	101M	=	197	293	GAAAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAA	FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP
A00147:108:HLLJFDMXX:2:1420:13431:15796	163	TRINITY_DN5604_c0_g2_i1	5	42	101M	=	197	293	GAAAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAA	FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP
A00147:121:HLLVMDMXX:1:1258:5141:32377	163	TRINITY_DN5604_c0_g2_i1	5	42	101M	=	107	203	GAAAAACCGACAGGAGGAATTTCTTTGTTACCAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAA	FFFFF,FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:F:FFFFFFFF	AS:i:-1	XN:i:0	XM:i:1	XO:i:0	XG:i:0	NM:i:1	MD:Z:30A70	YS:i:0	YT:Z:CP
A00147:121:HLLVMDMXX:1:1259:7645:5838	163	TRINITY_DN5604_c0_g2_i1	5	42	101M	=	107	203	GAAAAACCGACAGGAGGAATTTCTTTGTTACCAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAA	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFF	AS:i:-1	XN:i:0	XM:i:1	XO:i:0	XG:i:0	NM:i:1	MD:Z:30A70	YS:i:0	YT:Z:CP
A00147:108:HLLJFDMXX:1:1351:27082:1705	163	TRINITY_DN5604_c0_g2_i1	6	42	101M	=	158	253	AAAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAAA	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP
A00147:108:HLLJFDMXX:1:1475:28926:32706	163	TRINITY_DN5604_c0_g2_i1	6	42	101M	=	158	253	AAAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAAA	FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP
A00147:121:HLLVMDMXX:1:2162:31385:26381	163	TRINITY_DN5604_c0_g2_i1	6	42	101M	=	158	253	AAAAACCGACAGGAGGAANTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAAA	FFFFFFFFFFFFFFFFFF#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:	AS:i:-1	XN:i:0	XM:i:1	XO:i:0	XG:i:0	NM:i:1	MD:Z:18T82	YS:i:0	YT:Z:CP
A00147:121:HLLVMDMXX:2:1425:28745:30639	163	TRINITY_DN5604_c0_g2_i1	7	42	101M	=	169	263	AAAACCGACAGGAGGAATTTCTTTGTTAACAACAAAAACTAATATATTTCGCATACCTGACAGACATGGTGACAGCGCCTCTGATGTTCGCCGAATTAAAG	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:101	YS:i:0	YT:Z:CP

Bowte2 commands to generate the BAM file:

  # Use bowtie2 and paired-end options
  # Uses settings specified for use with DETONATE
  # and for paired end reads when using DETONATE.
  ${programs_array[bowtie2]} \
  -x ${transcriptome_name} \
  -S ${transcriptome_name}.sam \
  --threads ${threads} \
  -1 ${R1_list} \
  -2 ${R2_list} \
  --sensitive \
  --dpad 0 \
  --gbar 99999999 \
  --mp 1,1 \
  --np 1 \
  --score-min L,0,-0.1 \
  --no-mixed \
  --no-discordant

  # Convert SAM to sorted BAM
  #
  ${programs_array[samtools_view]} \
  -b \
  ${transcriptome_name}.sam \
  | ${programs_array[samtools_sort]} \
  -m ${mem_per_thread} \
  --threads ${threads} \
  -o ${transcriptome_name}.sorted.bam \
  -

Thanks for any insight!