Warning message: No primary alignment found for reference sequence Chr9. This could mean that the entire chromosome Chr9 is reapeated.

[Sabrili]

Hi @mnshgl0110 and everyone :)

I'm getting this warning when running syri, even though the program still seems to run and produces a .out file which I can use to create a plot with plotsr. My question is are these warnings important/can I overlook them since I still get an output, or are these actually an issue? And if they are an issue, how could I resolve this?

I've dotplotted these genomes together and they seem to align correctly.

I've added more details below:

• I've been using minimap2 to create the .out files instead of nucmer, as nucmer hadn't been working for me.
• minimap2 seems to work fine I think?:
  minimap2 -ax asm5 -t 24 --eqx refgenome qrygenome > OAustLongSat.sam

Output:
[M::mm_idx_gen::10.4261.50] collected minimizers
[M::mm_idx_gen::11.4452.07] sorted minimizers
[M::main::11.4452.07] loaded/built the index for 12 target sequence(s)
[M::mm_mapopt_update::11.8532.03] mid_occ = 490
[M::mm_idx_stat] kmer size: 19; skip: 19; is_hpc: 0; #seq: 12
[M::mm_idx_stat::12.1292.01] distinct minimizers: 41893830 (86.29% are singletons); average occurrences: 2.158; average spacing: 9.985
[M::worker_pipeline::84.8426.28] mapped 12 sequences
[M::main] Version: 2.17-r941
[M::main] CMD: minimap2 -ax asm5 -t 24 --eqx refgenome qrygenome
[M::main] Real time: 85.098 sec; CPU: 533.158 sec; Peak RSS: 10.994 GB
module load samtools/1.13-gcc-10.3.0

samtools view -b OAustLongSat.sam > OAustLongSat.bam

• but then when I run syri, it throws these warnings:
  syri -c OAustLongSat.bam -r refgenome -q qrygenome -F B --prefix OAustLongSat --nc 24 --log INFO

Warnings:

Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr9. This could mean that the entire chromosome Chr9 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr4. This could mean that the entire chromosome Chr4 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr1. This could mean that the entire chromosome Chr1 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr2. This could mean that the entire chromosome Chr2 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr10. This could mean that the entire chromosome Chr10 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr8. This could mean that the entire chromosome Chr8 is reapeated.
Reading BAM/SAM file - WARNING - No primary alignment found for reference sequence Chr6. This could mean that the entire chromosome Chr6 is reapeated.
local_variation - WARNING - Finished syri

Even though the program still generates a .out file that I can use with plotsr:

Any help would be extremely appreciated!
Thanks so much and happy new year everyone!

[mnshgl0110]

The output looks OK. I guess, the warning might be happening because of large differences in the size of these genomes (which is maybe caused by repeats?). You can try plotting a dotplot using the genome alignments. If that too looks OK, then I would imagine that there are no issues.

[Sabrili]

Thank you so much as always @mnshgl0110! I will do this :)