NO_COOR reads not in a single block at the end 0 -1

Question

I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse "GCA_000001635.9_GRCm39_genomic.fna".

Apparently everything works well up to here. Now I'm using samtools 1.9 to convert sam to bam, to sort those bam and create the index, just like this:

samtools view -bS ${file} > ${file}.bam

samtools sort -n ${file} -o ${file}_sorted.bam

samtools index  ${file}_sorted.bam

But when indexing I get this error:

[bam_sort_core] merging from 11 files and 1 in-memory blocks... [E::hts_idx_push] NO_COOR reads not in a single block at the end 0 -1 samtools index: failed to create index for "CD4_1A_concat.bam_sorted.bam": No such file or directory

[bam_sort_core] merging from 11 files and 1 in-memory blocks... [E::hts_idx_push] NO_COOR reads not in a single block at the end 23 -1 samtools index: failed to create index for "CD4_1B_concat.bam_sorted.bam": No such file or directory [bam_sort_core] merging from 11 files and 1 in-memory blocks...

And more...

Any help would be appreciated!

Answer 1

This is essentially a duplicate of Samtools Index: Chromosome Blocks not Continuous but with different error messages, and a duplicate of Error given while trying to index a BAM file with Samtools Index - NO COOR?.

samtools sort -n ${file} -o ${file}_sorted.bam

These error messages also indicate that the reads are not sorted by coordinate — in particular, that the unplaced unmapped reads with RNAME * are not all together at the end of the file.

This is because samtools sort -n has been used to sort the reads by name instead. Remove -n to sort by position, which is what is needed to prepare a BAM file for indexing with samtools index.