Inconsistent Genome Representation in Graph Construction: Variants Concentrated on a Single Chromosome

[HuangYihang1222]

Hello,

I have been working constructing a graph-based pangenome using minigraph-cactus with three genomes—genome A is set as the reference, and B1 and B2 represent the two haplotypes of the same individual—to construct a graph and map second-generation sequencing data back to this graph for variant calling. While both the graph construction and mapping processes appear to work fine, I encountered an issue: most of the detected variants are concentrated on a single chromosome.

Upon inspecting the constructed graph, I noticed that on this particular chromosome’s W-line, all three genome names (A, B1, and B2) are present. In contrast, on the other chromosomes, either only genome A is present or genome A and one other genome appear.

Could you please advise on how I might resolve this issue, and what could be the potential causes behind it?

Thank you very much for your time and assistance.

Best regards,
Yihang Huang

[glennhickey]

I guess the place to start is in chrom-subproblems/minigraph.split.log. It gives a list of the reference chromosome assignment for each input contig. Contigs considered "ambiguous" don't make it into the final graph.

[HuangYihang1222]

Thank you for your suggestion. I checked my log file, and its content is as follows:

Query contig is ambiguous: id=NA.2|Hic_asm_18  len=55506995 cov=0.222502 (vs 0.25) uf= infinity (vs 2)
Assigned contig to Hic_asm_14: id=NA.2|Hic_asm_14  len=60512409 cov=0.256855 (vs 0.25) uf=482.279 (vs 2)
 Reference contig mappings:
  Hic_asm_8: 32228
  Hic_asm_14: 15542893
Query contig is ambiguous: id=NA.2|Hic_asm_16  len=61691755 cov=0.163628 (vs 0.25) uf= infinity (vs 2)
Assigned contig to Hic_asm_10: id=NA.2|Hic_asm_10  len=44211056 cov=0.29647 (vs 0.25) uf=762.938 (vs 2)
 Reference contig mappings:
  Hic_asm_8: 17180
  Hic_asm_10: 13107267
Assigned contig to Hic_asm_8: id=NA.2|Hic_asm_8  len=53910277 cov=0.277231 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.2|Hic_asm_7  len=49669858 cov=0.078777 (vs 0.25) uf=324.26 (vs 2)
 Reference contig mappings:
  Hic_asm_4: 12067
  Hic_asm_7: 3912840
  Hic_asm_14: 5041
Query contig is ambiguous: id=NA.2|Hic_asm_6  len=44461152 cov=0.159471 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_17  len=72449833 cov=0.212237 (vs 0.25) uf=496.082 (vs 2)
 Reference contig mappings:
  Hic_asm_8: 30996
  Hic_asm_17: 15376569
Query contig is ambiguous: id=NA.1|Hic_asm_16  len=58521165 cov=0.149565 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_15  len=72096067 cov=0.152389 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_13  len=63683632 cov=0.155302 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_12  len=45018532 cov=0.105382 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_13: id=CN|Hic_asm_13  len=64317445 cov=0.999916 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.2|Hic_asm_2  len=55085163 cov=0.177952 (vs 0.25) uf= infinity (vs 2)
Assigned contig to Hic_asm_1: id=NA.2|Hic_asm_1  len=58035721 cov=0.284337 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_4: id=CN|Hic_asm_4  len=48848854 cov=0.999994 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.2|Hic_asm_17  len=72311999 cov=0.226457 (vs 0.25) uf=518.772 (vs 2)
 Reference contig mappings:
  Hic_asm_9: 31566
  Hic_asm_14: 15050
  Hic_asm_17: 16375566
Query contig is ambiguous: id=NA.2|Hic_asm_13  len=61943115 cov=0.229907 (vs 0.25) uf=1420 (vs 2)
 Reference contig mappings:
  Hic_asm_9: 2676
  Hic_asm_10: 4301
  Hic_asm_13: 14241186
  Hic_asm_14: 10029
Assigned ref-contig to Hic_asm_12: id=CN|Hic_asm_12  len=41146388 cov=0.999994 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_14  len=62034392 cov=0.195481 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_10: id=CN|Hic_asm_10  len=44185251 cov=0.999602 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_8: id=CN|Hic_asm_8  len=45199631 cov=1 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_18  len=56271462 cov=0.188452 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_17: id=CN|Hic_asm_17  len=56107779 cov=0.999993 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_11: id=CN|Hic_asm_11  len=60793815 cov=0.999992 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_7: id=CN|Hic_asm_7  len=45903895 cov=0.999849 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_2: id=CN|Hic_asm_2  len=53936972 cov=0.999997 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.2|Hic_asm_15  len=71854087 cov=0.194147 (vs 0.25) uf=1074.25 (vs 2)
 Reference contig mappings:
  Hic_asm_4: 12986
  Hic_asm_14: 123
  Hic_asm_15: 13950253
Query contig is ambiguous: id=NA.2|Hic_asm_5  len=60716626 cov=0.110871 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_3: id=CN|Hic_asm_3  len=72978263 cov=0.999997 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_14: id=CN|Hic_asm_14  len=59003186 cov=0.999632 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_9: id=CN|Hic_asm_9  len=53416110 cov=0.999997 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.2|Hic_asm_11  len=66124883 cov=0.168124 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_5: id=CN|Hic_asm_5  len=55474205 cov=0.999996 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_1: id=CN|Hic_asm_1  len=57770234 cov=0.999999 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_11  len=67187999 cov=0.149641 (vs 0.25) uf=1214.26 (vs 2)
 Reference contig mappings:
  Hic_asm_1: 5543
  Hic_asm_11: 10054059
  Hic_asm_17: 8280
Assigned ref-contig to Hic_asm_15: id=CN|Hic_asm_15  len=74437777 cov=0.999771 (vs 0.25) uf= infinity (vs 2)
Assigned contig to Hic_asm_9: id=NA.2|Hic_asm_9  len=52489210 cov=0.268502 (vs 0.25) uf=2901.08 (vs 2)
 Reference contig mappings:
  Hic_asm_9: 14093466
  Hic_asm_14: 4858
Assigned ref-contig to Hic_asm_16: id=CN|Hic_asm_16  len=72684925 cov=0.999923 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_18: id=CN|Hic_asm_18  len=37903052 cov=0.999991 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_1  len=58073023 cov=0.188999 (vs 0.25) uf=126.077 (vs 2)
 Reference contig mappings:
  Hic_asm_1: 10975753
  Hic_asm_8: 87056
Query contig is ambiguous: id=NA.2|Hic_asm_4  len=49691924 cov=0.154414 (vs 0.25) uf=87194.8 (vs 2)
 Reference contig mappings:
  Hic_asm_4: 7673139
  Hic_asm_15: 88
Query contig is ambiguous: id=NA.1|Hic_asm_5  len=59565153 cov=0.0748572 (vs 0.25) uf=182.748 (vs 2)
 Reference contig mappings:
  Hic_asm_5: 4458878
  Hic_asm_16: 24399
Query contig is ambiguous: id=NA.1|Hic_asm_2  len=65108644 cov=0.145186 (vs 0.25) uf=7238.01 (vs 2)
 Reference contig mappings:
  Hic_asm_2: 9452836
  Hic_asm_15: 1306
Query contig is ambiguous: id=NA.1|Hic_asm_3  len=67786512 cov=0.109125 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_10  len=54534567 cov=0.153023 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_4  len=49799525 cov=0.136897 (vs 0.25) uf=980.78 (vs 2)
 Reference contig mappings:
  Hic_asm_4: 6817401
  Hic_asm_13: 6951
  Hic_asm_18: 2038
Query contig is ambiguous: id=NA.2|Hic_asm_12  len=43698553 cov=0.126797 (vs 0.25) uf=335.199 (vs 2)
 Reference contig mappings:
  Hic_asm_6: 16530
  Hic_asm_9: 2089
  Hic_asm_12: 5540843
  Hic_asm_17: 10359
Query contig is ambiguous: id=NA.2|Hic_asm_3  len=66872696 cov=0.0999454 (vs 0.25) uf=8794.24 (vs 2)
 Reference contig mappings:
  Hic_asm_3: 6683621
  Hic_asm_8: 760
Query contig is ambiguous: id=NA.1|Hic_asm_7  len=47551965 cov=0.0839781 (vs 0.25) uf= infinity (vs 2)
Assigned ref-contig to Hic_asm_6: id=CN|Hic_asm_6  len=45146159 cov=0.999989 (vs 0.25) uf= infinity (vs 2)
Query contig is ambiguous: id=NA.1|Hic_asm_6  len=45402704 cov=0.144119 (vs 0.25) uf=394.846 (vs 2)
 Reference contig mappings:
  Hic_asm_6: 6543385
  Hic_asm_8: 9339
  Hic_asm_16: 16572
  Hic_asm_17: 4341
Assigned contig to Hic_asm_8: id=NA.1|Hic_asm_8  len=54440431 cov=0.25957 (vs 0.25) uf=725.789 (vs 2)
 Reference contig mappings:
  Hic_asm_8: 14131113
  Hic_asm_9: 19470
  Hic_asm_15: 953
Query contig is ambiguous: id=NA.1|Hic_asm_9  len=52313486 cov=0.171517 (vs 0.25) uf=389.523 (vs 2)
 Reference contig mappings:
  Hic_asm_8: 23035
  Hic_asm_9: 8972654
  Hic_asm_14: 5808

I noticed that many of the haplotype chromosomes I provided were considered as "ambiguous." Could this be due to issues with genome assembly quality?