Hi there, I seem to be getting this error when reading in a BAM file which was generated by PBMM2 align on pacbio data. I have tried to google the error message but there are no results. I wonder if anyone has ideas on what the error might be? Thanks in advance
ERROR: sequence length in the BAM record is out of the expected region
Here is the code
library(Rsubread)
mycounts<-featureCounts("test.bam",annot.ext="test.gtf", isGTFAnnotationFile=TRUE, isPaired End=FALSE,allowMultiOverlap=T,GTF.featureType=c("transcript"),isLongRead=T,useMe taFeatures=F,countMultiMappingReads=T,reportReads="CORE",minFragLength="100",max FragLength="10000")
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.0
//========================== featureCounts setting ===========================\\
|| ||
|| Input files : 1 BAM file ||
|| ||
|| test.bam ||
|| ||
|| Paired-end : no ||
|| Count read pairs : no ||
|| Annotation : test.gtf (GTF) ||
|| Dir for temp files : . ||
|| Assignment details : <input_file>.featureCounts ||
|| (Note that files are saved to the output directory) ||
|| ||
|| Threads : 1 ||
|| Level : feature level ||
|| Multimapping reads : counted ||
|| Multi-overlapping reads : counted ||
|| Min overlapping bases : 1 ||
|| Long read mode : yes ||
|| ||
\\============================================================================//
//================================= Running ==================================\\
|| ||
|| Load annotation file test.gtf ... ||
|| Features : 267787 ||
|| Meta-features : 67977 ||
|| Chromosomes/contigs : 440 ||
|| ||
|| Process BAM file test.bam... ||
ERROR: sequence length in the BAM record is out of the expected region: 1467, 81 5
|| Single-end reads are included. ||
|| Total alignments : 1180654 ||
|| Successfully assigned alignments : 1177384 (99.7%) ||
|| Running time : 0.26 minutes ||
|| ||
|| Write the final count table. ||
|| Write the read assignment summary. ||
|| ||
\\============================================================================//
•
written
by
elengss •
1 answer
I think you should not bother too much. The important information is Successfully assigned alignments : 1177384 (99.7%).
As far as the error is concerned, I would presume that you have a read that maps to a contig that is shorter than the read itself. What is the shortest length of reference contigs? Do you have a contig that is just 81bp long? In that case, I would presume you have a read of the length 1467 mapping to a contig of 81 bases. (Or maybe also being 81 bases longer than the contig?)
To know for sure, you would need to download the source-code of subread and then search for that error message. I usually do something like
grep "sequence length in the BAM record" -R src -C 5
Just replace src with the path to the actual directory of the code.
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