Unexpected NULL genotypes in some multisample calling

Hi,

I’ve been using sniffles with content for quite a while now. But just yesterday an unexpected pheomenon occurred for a sample that I’d like to report, since I couldn’t find out the reason.

I’m calling SVs using sniffles’ multisample functionality as follows:

sniffles --input tumor.bam --snf tumor.snf  # step 1
sniffles --input normal.bam --snf normal.snf  # step 2
sniffles --input tumor.snf normal.snf --vcf tumor_normal.vcf  # step 3

This approach had no issues; for the majority of the samples I get something like the following which I have no problem with.

However, I noticed that for one sample pair, all the genotypes of one of the samples (with the tumor header) only have ./.:0:0:0:NULL. I’d like to add that such genotyping was done for the tumor.snf when its VCF was created separately by sniffles --input tumor.snf --vcf tumor.vcf.

Hope I’ve just done something wrong and it’s a quick fix, but in case it’s not, I’ve pasted the output of three log files from step 1 to 3 (commented above).

[LOG] sniffles --input tumor.bam --snf tumor.snf # step 1

Running Sniffles2, build 2.0.7
  Run Mode: call_sample
  Start on: 2022/08/24 14:32:17
  Working dir: /juno/work/shah/users/chois7/ONT/sniffles
  Used command: /home/chois7/chois7/envs/sniffles/bin/sniffles --input /juno/work/shah/isabl_data_lake/analyses/60/35/26035/results/minimap2/SHAH_H000019_T10_01_WG02_R1.sorted.bam --snf results/SPECTRUM-OV-022/tumor.snf
==============================
Opening for reading: /juno/work/shah/isabl_data_lake/analyses/60/35/26035/results/minimap2/SHAH_H000019_T10_01_WG02_R1.sorted.bam
Opening for writing: results/SPECTRUM-OV-022/tumor.snf

Analyzing 19707267 alignments total...

  985792/19707267 alignments processed (5%, 3564/s); 3113/3366 tasks done; parallel 4/4; 8445 candidates.
 2059384/19707267 alignments processed (10%, 4696/s); 3344/3366 tasks done; parallel 4/4; 40028 candidates.
 3121946/19707267 alignments processed (15%, 5501/s); 3347/3366 tasks done; parallel 4/4; 131008 candidates.
 4042568/19707267 alignments processed (20%, 5038/s); 3349/3366 tasks done; parallel 4/4; 217538 candidates.
 5487256/19707267 alignments processed (27%, 6549/s); 3351/3366 tasks done; parallel 4/4; 352173 candidates.
 6375459/19707267 alignments processed (32%, 6400/s); 3353/3366 tasks done; parallel 4/4; 439481 candidates.
 7437291/19707267 alignments processed (37%, 6458/s); 3355/3366 tasks done; parallel 4/4; 542896 candidates.
 9003836/19707267 alignments processed (45%, 6677/s); 3357/3366 tasks done; parallel 4/4; 705956 candidates.
 9915059/19707267 alignments processed (50%, 7030/s); 3358/3366 tasks done; parallel 4/4; 785408 candidates.
11582078/19707267 alignments processed (58%, 6686/s); 3360/3366 tasks done; parallel 4/4; 960122 candidates.
12888637/19707267 alignments processed (65%, 7252/s); 3361/3366 tasks done; parallel 4/4; 1091829 candidates.
13880804/19707267 alignments processed (70%, 7333/s); 3362/3366 tasks done; parallel 4/4; 1198999 candidates.
19707267/19707267 alignments processed (100%, 7711/s); 3366/3366 tasks done; parallel 0/4; 1737670 candidates.
Took 2555.44s.

Done.
Wrote 1737670 SV candidates to results/SPECTRUM-OV-022/tumor.snf (for multi-sample calling).

[LOG] sniffles --input normal.bam --snf normal.snf # step 2

Running Sniffles2, build 2.0.7
  Run Mode: call_sample
  Start on: 2022/08/24 14:33:58
  Working dir: /juno/work/shah/users/chois7/ONT/sniffles
  Used command: /home/chois7/chois7/envs/sniffles/bin/sniffles --input /juno/work/shah/isabl_data_lake/analyses/69/95/26995/results/minimap2/SHAH_H000019_N04_01_WG01_R1.sorted.bam --snf results/SPECTRUM-OV-022/normal.snf
==============================
Opening for reading: /juno/work/shah/isabl_data_lake/analyses/69/95/26995/results/minimap2/SHAH_H000019_N04_01_WG01_R1.sorted.bam
Opening for writing: results/SPECTRUM-OV-022/normal.snf

Analyzing 11540540 alignments total...

  681697/11540540 alignments processed (5%, 16679/s); 62/84 tasks done; parallel 4/4; 17873 candidates.
 1309793/11540540 alignments processed (11%, 16782/s); 65/84 tasks done; parallel 4/4; 68578 candidates.
 2101645/11540540 alignments processed (18%, 21760/s); 67/84 tasks done; parallel 4/4; 138118 candidates.
 2812627/11540540 alignments processed (24%, 20889/s); 69/84 tasks done; parallel 4/4; 189170 candidates.
 3373870/11540540 alignments processed (29%, 23904/s); 71/84 tasks done; parallel 4/4; 241333 candidates.
 4121555/11540540 alignments processed (35%, 19471/s); 73/84 tasks done; parallel 4/4; 311150 candidates.
 5018521/11540540 alignments processed (43%, 21906/s); 75/84 tasks done; parallel 4/4; 387484 candidates.
 5914753/11540540 alignments processed (51%, 24614/s); 76/84 tasks done; parallel 4/4; 428327 candidates.
 6625555/11540540 alignments processed (57%, 23017/s); 77/84 tasks done; parallel 4/4; 472048 candidates.
 7227317/11540540 alignments processed (62%, 22957/s); 78/84 tasks done; parallel 4/4; 521145 candidates.
 7757073/11540540 alignments processed (67%, 23746/s); 79/84 tasks done; parallel 4/4; 573392 candidates.
 8337877/11540540 alignments processed (72%, 23989/s); 80/84 tasks done; parallel 4/4; 627228 candidates.
11540540/11540540 alignments processed (100%, 24590/s); 84/84 tasks done; parallel 0/4; 886934 candidates.
Took 469.32s.

Done.
Wrote 886934 SV candidates to results/SPECTRUM-OV-022/normal.snf (for multi-sample calling).

[LOG] sniffles --input tumor.snf normal.snf --vcf tumor_normal.vcf # step 3

Running Sniffles2, build 2.0.7
  Run Mode: combine
  Start on: 2022/08/24 15:37:49
  Working dir: /juno/work/shah/users/chois7/ONT/sniffles
  Used command: /home/chois7/chois7/envs/sniffles/bin/sniffles --input /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/tumor.snf /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/normal.snf --vcf /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/tumor_normal.vcf
==============================
Opening for writing: /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/tumor_normal.vcf (multi-sample, sorted)
Verified headers for 2 .snf files.
The following samples will be processed in multi-calling:
    /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/tumor.snf (sample ID in output VCF='tumor')
    /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/normal.snf (sample ID in output VCF='normal')

Calling SVs across 2 samples (2624604 candidates total)...

 138118/2624604 candidates processed (5%, 15986/s); 67/84 tasks done; parallel 4/4; 4936 SVs.
 270765/2624604 candidates processed (10%, 23090/s); 72/84 tasks done; parallel 4/4; 9174 SVs.
 428327/2624604 candidates processed (16%, 29292/s); 76/84 tasks done; parallel 4/4; 14014 SVs.
 573392/2624604 candidates processed (21%, 31763/s); 79/84 tasks done; parallel 4/4; 18490 SVs.
 685427/2624604 candidates processed (26%, 32021/s); 81/84 tasks done; parallel 3/4; 21521 SVs.
 886934/2624604 candidates processed (33%, 39118/s); 84/84 tasks done; parallel 0/4; 26806 SVs.
Took 22.67s.

Done.
Wrote 26806 called SVs to /juno/work/shah/users/chois7/ONT/sniffles/results/SPECTRUM-OV-022/tumor_normal.vcf (multi-sample, sorted)

[soymintc]

@fritzsedlazeck I’ve checked the logs and the original bam files to find the cause, and this seems to be because the tumor and normal bam files for the sample in question were aligned to different reference genomes. Sorry for the confusion; I’ll close the issue. 😅

[fritzsedlazeck]

Thanks for reporting this. Indeed a few entries can have ./. because of low coverage. Did you find anything strange in the VCF file of that tumor sample ? Like does it have SV reported etc ?

I would first try to rerun the tumor sample and check if the output files have a size (snf file) .

Thanks Fritz