How to extract all sequences mapped to a transcript from Kallisto output

Question

[hzc363]

I ran Kallisto with the --pseudobam option. How do I extract all the short reads that are mapped to a single transcript (e.g. ENST00000367969.8)? As a person without any previous SAM/BAM experience, I tried the following things without success. Really appreciate some help here.

I first tried:

samtools view -S pseudoalignments.bam ENST00000367969.8

but got the following error:

[E::idx_find_and_load] Could not retrieve index file for 'pseudoalignments.bam' [main_samview] random alignment retrieval only works for indexed BAM or CRAM files.

I tried to index the file using:

samtools index pseudoalignments.bam

but get the following error:

[E::hts_idx_push] NO_COOR reads not in a single block at the end 79729 -1 
[E::sam_index] Read 'A00742:184:HLTF2DSXY:4:1101:4417:1000' with ref_name='ENST00000556431.1', ref_length=4142, flags=83, pos=2098 cannot be indexed samtools index: failed to create index for "pseudoalignments.bam"

[Ram]

Cross-posted on bioinfo SE: https://bioinformatics.stackexchange.com/q/17685/650

Don't post your question on mutliple sites. That's inconsiderate, like asking multiple people to help you with something just to ensure you get help faster.

Answer 1

[Shred]

Before indexing you need to sort your alignment

samtools sort -o pseudoalignment