The SAM/BAM file declares less than one reference sequence!

[Heterosigma]

Hi, I tried rsem-calculate-expression using bowtie, and the mapping step appeared to be completed. Then I got the error message

"The SAM/BAM file declares less than one reference sequence!"

since the BAM file is created in the *.temp directory , it appears strange to me.
When I check the *.stat directory, it is empty.

Also, the last several lines of the output say

bowtie -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -p 8 -a -m 200 -S task00831_Trinity3 -1 DNBSEQ_Haka1_012023_Read1.fq -2 DNBSEQ_Haka1_012023_Read2.fq 2> DNBSEQ_Haka1_012023.log | samtools view -b -o DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023.bam -

rsem-parse-alignments task00831_Trinity3 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023 DNBSEQ_Haka1_012023.stat/DNBSEQ_Haka1_012023 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023.bam 3 -tag XM
"rsem-parse-alignments task00831_Trinity3 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023 DNBSEQ_Haka1_012023.stat/DNBSEQ_Haka1_012023 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

Here, the task00831_Trinity3.fasta file was the original reference file, and the rsem-prepare-references was completed by

rsem-prepare-reference --bowtie --gff3 task00831_Trinity3.gff3 -p 20 --bowtie task00831_Trinity3 task00831_Trinity3

and the files with several different extensions were created.

Could somebody tell me what I did wrong??

When I try

rsem-parse-alignments task00831_Trinity3 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023.bam DNBSEQ_Haka1_012023.stat/DNBSEQ_Haka1_012023 DNBSEQ_Haka1_012023.temp/DNBSEQ_Haka1_012023.bam

I got the same message,
"The SAM/BAM file declares less than one reference sequence!"

[Threeleben]

I got the same problem, have u solved it?

[albustruong]

Hi, I got this error once. But I found out that it had nothing to do with the indexed reference (I did try creating a new indexed reference). Instead, the problem lied in my rsem-calculate-expression arguments:

Trouble-causing script

rsem-calculate-expression
--bowtie2 --bowtie2-path /usr/bin/bowtie2 --phred33-quals
--paired-end
--p 6 --output-genome-bam --sort-bam-by-coordinate
sub1-Ca13mCh-LGC9389_L1_1_cleaned.fq.gz
sub1-Ca13mCh-LGC9389_L1_2_cleaned.fq.gz
human
sub1-Ca13mCh-LGC9389_L1_PE_quals
2>&1 | tee sub1-Ca13mCh-LGC9389_L1_log.txt

I just removed the --bowtie2-path flag and it worked.

New script

rsem-calculate-expression
--bowtie2 --phred33-quals
--paired-end
--p 6 --output-genome-bam --sort-bam-by-coordinate
sub1-Ca13mCh-LGC9389_L1_1_cleaned.fq.gz
sub1-Ca13mCh-LGC9389_L1_2_cleaned.fq.gz
human
sub1-Ca13mCh-LGC9389_L1_PE_quals
2>&1 | tee sub1-Ca13mCh-LGC9389_L1_log.txt

Hope this helps.