Error in creating BAM index

Question

[Constantine]

Hello

I've created an alignment (SAM and subsequently BAM file) using samtools. However, when I'm trying to create an index out of the BAM file I get the following message:

$ samtools index alignment.new.bam
[E::hts_idx_push] NO_COOR reads not in a single block at the end 15 -1

I have no clue what that means so any help would be much appreciated

Thanks

[GouthamAtla]

Can you paste the commands used for alignment and sam --> bam ?

[Constantine]

For the alignment:

$ bowtie2 -p 20 -x mm10 -U SRR1557699_1.fastq.gz -S alignment.new.sam

For Sam -> Bam

$ samtools view -bS alignment.new.sam > alignment.new.bam

Answer 1

[Zev.Kronenberg]

I cannot tell from the post if you have sorted you bam file. You need to sort bam files before indexing them.

[Dan D]

You've almost certainly identified the problem. The error message is a complaint that the unaligned reads are not at the end of his BAM.

[Constantine]

Thanks. That solved the problem :)

[behrimg]

So, I am encountering a similar problem.

The message I receive is

[E::hts_idx_push] NO_COOR reads not in a single block at the end 0 -1

However I am not receiving this message for all samples. The script is a loop so all my samples are being treated the same, yet 3 made it through without error and three did not.

Here are my commands for reference.

samtools view -bS -T REFGENOME.fa SAMPLE1.sam > Sample1.bam
samtools sort SAMPLE1.bam SAMPLE1.sorted
samtools index SAMPLE1.sorted.bam

From here the data is passed to picard/GATK

Also just a note, my reference is E. coli so the problem isn't chromosome size limit.

Answer 2

[ks]

Also, the sorted file needs to be coordinate sorted, not by feature name (don't include the -n parameter).