The content is too long, I have to cut it in two.

1. All the variants in my input VCF are biallelic. I did not perform any augmentation before calling, so I would expect that the output VCF should not contain any new POS or genotypes. However, in reality, the output VCF contains many multi-allelic variants. For instance, the variant mentioned in the previous example appears as a multi-allelic variant in sample C, whereas the input VCF indicates that it is biallelic.

   sample C:
   NC_058080.1_1   59145   >89127697>89127735  ATAAATGGTAGAAAAATTGCAACAGGTTTTATTGAAAAAAAAACACTGGCTCAATTGGACATCACTATGATCATTTGCACTGGTATAAACGGTAGAAAAATTGCAACAGGTTTTTGAGAAACGGCAAAATTCCCAATTACATGTTCCCTGAATGCGGCAAAACTGTGCGCTGCTACTGATGCACTAAAATACACTTATTTGTGCATCTAAAATGTAGAAAAATTGCAACAGGTTTTAGGGAAACAGAAATGCACAATTGGACATCACCTGAGTGCATCCAAACTATACACTGTTATTGACGCACTTATAAATGGTAGAAAAATTGAAATAAGTTTTGAGAAACACCAAAATTTCAATAGGACGACCTCTGGGTGAAACAAAAATATACACTATTATGCTGCTGTTAATGCAGTAAAATATGATTATTGCACATATATAAAATTTTGAAAAATTGTAAGGCTGGCTTCTGTACAGTCTGTTGTCTTCTTAAGGATCTCTGTGCTGCAGTAGTCTGGACTAGGGCTCAGCTAAGGGGAGGCCTGCATGATTAGGGTTGGTTGTGAGGGTCAGGGAGGTTAGAGGTTAAGGGATTTGAATGTCTAGTAAAAGAGGGGGGGCATCGCATAGGAGAGGAGTTTGGGGGGCTATTTATGGGGGTGTCTTGTAGTGAGGGGTGCCATTTTGTGCACTGAGAAAAACTGGTAAGATGTCCCAGACACTTACCATCAATGATATGCTGGCTAGGCTAAGAGAGGCAGCGGCGGAGCGGGGCCATGAGTGGCTGAGCTCCCAGATGTCCGCCATATTGAGGGCTGAGGCAGCGGGTACGGTTAGCTCGCCTCCGGAAGGAAGACGGACATGGCTGGTACGGCCGCCCGCGCGCCTGAGTCCCAGTGAGACCCCCCGAGTTCGGCGCCGTGTCAGGAGCCCCTCCGGGGACCCTCCACCTCGGGGGAATGCAGGGCAGACTACATCCTCATCCTCCTGGCGTGGGAGGAATCCATGTGGCAGGCGTAACCCAGCGCAGGGCAGGAGGGCCCTCCCCTGCCCTCTCCGGTGAGCAGCGATGGATCGGGAGCGGCGGGATCCTTACCTTCTAGTGGGGTCAGGAGGCCTGCTCAGCGAGGGATGGACGGGAGGCATGTGGCACGCGAGG    A,ATAAATGGTAGAAAAATTGCAACAGGTTTTATTGAAAAAAAAACACTGGCTCAATTGGACATCACTATGATCATTTGCACTGGTATAAACGGTAGAAAAATTGCAACAGGTTTTTGAGAAACGGCAAAATTCCCAATTACATGTTCCCTGAATGCGGCAAAACTGTGCGCTGCTACTGATGCACTAAAATACACTTATTTGTGCATCTAAAATGTAGAAAAATTGCAACAGGTTTTAGGGAAACAGAAATGCACAATTGGACATCACCTGAGTGCATCCAAACTATACACTGTTATTGACGCACTTATAAATGGTAGAAAAATTGAAATAAGTTTTGAGAAACACCAAAATTTCAATAGGACGACCTCTGGGTGAAACAAAAATATACACTATTATGCTGCTGTTAATGCAGTAAAATATGATTATTGCACATATATAAAATTTTGAAAAATTGTAAGGCTGGCTTCTGTACAGTCTGTTGTCTTCTTAAGGATCTCTGTGCTGCAGTAGTCTGGACTAGGGCTCAGCTAAGGGGAGGCCTGCATGATTAGGGTTGGTTGTGAGGGTCAGGGAGGTTAGAGGTTAAGGGATTTGAATGGTCTTGTAGTGAGGGGTGCCATTTTGTGCACTGAGAAAAACTGGTAAGATGTCCCAGACACTTACCATCAATGATATGCTGGCTAGGCTAAGAGAGGCAGCGGCGGAGCGGGGCCATGAGTGGCTGAGCTCCCAGATGTCCGCCATATTGAGGGCTGAGGCAGCGGGTACGGTTAGCTCGCCTCCGGAAGGAAGACGGACATGGCTGGTACGGCCGCCCGCGCGCCTGAGTCCCAGTGAGACCCCCCGAGTTCGGCGCCGTGTCAGGAGCCCCTCCGGGGACCCTCCACCTCGGGGGAATGCAGGGCAGACTACATCCTCATCCTCCTGGCGTGGGAGGAATCCATGTGGCAGGCGTAACCCAGCGCAGGGCAGGAGGGCCCTCCCCTGCCCTCTCCGGTGAGCAGCGATGGATCGGGAGCGGCGGGATCCTTACCTTCTAGTGGGGTCAGGAGGCCTGCTCAGCGAGGGATGGACGGGAGGCATGTGGCACGCGAGG  22.2838 PASS    AT=>89127697>89127698>89127699>89127700>89127701>89127702>89127703>89127704>89127705>89127706>89127707>89127708>89127709>89127710>89127711>89127712>89127713>89127714>89127715>89127716>89127717>89127718>89127719>89127720>89127721>89127722>89127723>89127724>89127725>89127726>89127727>89127728>89127729>89127730>89127731>89127732>89127733>89127734>89127735,>89127697>89127735,>89127697>89127698>89127699>89127700>89127701>89127702>89127703>89127704>89127705>89127706>89127707>89127708>89127709>89127710>89127711>89127712>89127713>89127714>89127715>89127716>89127719>89127720>89127721>89127722>89127723>89127724>89127725>89127726>89127727>89127728>89127729>89127730>89127731>89127732>89127733>89127734>89127735;DP=7    GT:DP:AD:GL:GQ:GP:XD:MAD    1/2:7:1,1,5:-4.29935,-3.60807,-3.08519,-10.3865,-3.60807,-4.29935:6:-1.28391:12.2142:1
   

   Why is this happening? are these multiallelic reliable?

2. I would like to hear your opinion on whether to enable nested calling mode. Based on my understanding, using short-read data to call nested variants may increase the false positive rate (inaccurate calling). If my goal is to have the most accurate calling possible and I don't mind sacrificing the quantity of calls (some inside variants not being called), then I should not enable it.

Thanks for your attention.

Maxine

I failed with the command "vg call -v." All the variants from -v VCF file failed to be called. I suspect it might be because I constructed VG for each chromosome following the tutorial: Working with a Whole Genome Variation Graph

Anyway, I'll provide the command and some of the VG call warning messages below, hoping that this information is sufficiently detailed:

construction for each chromsome:
    vg construct -f -S -a \
    -t ${SLURM_CPUS_PER_TASK} \
    -R ${chrom} \
    -r ${ref_genome} \
    -v ${input_vcf} \
    > ${chrom}.${vg_pre}.vg

Note ID coordination:
vg ids -j $(cat ${chrom_list} | while read chrom; do echo "${chrom}.${vg_pre}.vg"; done)

index for xg:
vg index -t ${SLURM_CPUS_PER_TASK} -p \
        -x ${vg_pre}.16in1.xg \
        $(cat ${chrom_list} | while read chrom; do echo "${chrom}.${vg_pre}.vg"; done)

pruned vg then index for gcsa:
vg index -t ${SLURM_CPUS_PER_TASK} -p \
    --temp-dir ${temp_path} \
    -g ${vg_pre}.16in1.gcsa \
    $(cat ${chrom_list} | while read chrom; do echo "pruned.${chrom}.${vg_pre}.vg"; done) 

vg call:
  vg call ${xg_file} \
    -k ${pack_file} \
    -r ${snarls_file} \
    -t ${SLURM_CPUS_PER_TASK} \
    -v ${input_vcf} \
    > ${output_vcf}
----------------------------------------------
most variants calling failed like this:
[VCFTraversalFinder] Warning: No alt path (prefix=_alt_e025a9b83d893c574eeab3a2991ab82e907c6e60_) found in graph for variant.  It will be ignored:
NC_008410.1 13489   Sniffles2.INS.9M2EF T   CATTTGGATTTGAAAGCAGCCGCATGATACTGACCATTTTGTCGACGTTGTTTGGACTCTTCTTATATGTCTCAAATTTTACTGATGAGGCTCTTTTTTCTTAGTATAATTAGTACTAATGACTTCCAAATCATAAGAATTTTCTTGGTTAAACCCCGAGAGAAAATAATGACCTATATTGTTCTTCTTACCTTAATATTGTTCAGTTTTAGCCTCATCAGCTTCTGACTACCAACTATTAGCTCGGCTCAGAAAAACTCTCACCTTATGAGTGCGGCTTTGACCCGCTAGGATCCGCCCGCCTCCCATATTCCATGCGGTTCTTTCTAGTGCTATTCTTTTTCTCCTCTTTGACCTAGAAAATTGCCCTCCTCTCCCCACCCCCTGAGCTGCACAACTTCCCTATCCCACCCGGTCTATCTTCTTGCTCAGTAATCCTAATTCTTTTAACCTTAGGGTTTGTCTATGAGTGACTTCAAGGAGGCCTGAGAATGAGCTGAATAAGGAGTTAGTTCTTAAAAAAGACAGCTGATTTCGACTCAGCAAAATTATGGTTTAACCCCATAGCGCCTTTTGATAACAAACGAATCCTGATTACTCTCTTCCACATTTATATTGAGCCTATTGGCCTGTCATTTCACCGGGCGCCCTGCTCTCAGCCCTGCTCTGCCTAGGAGGGCATGATACTCTCAAAATCTTCATTGGCCTGGCCTCTGGGCCTAAAAATTTGCTTAATCGCCCCCCTAATGTACCTATTGCCATGTAAAACTATGTCTGCATGTGAGGCGGGCTCGGCCTCTCCCTCATTAATCGCCACTGCTCGAACCCACGGCTCAGATAATTTGAATACCTTAAACCTCCTGCAATGCTAATAATATTATCCCTCTTGCCACCCTCCCTCCTCTCAACCTGACTGGCTCCCTTCAAAGCGGTTGTGAGAAATTATCACAACCCAGACCTTAGTTTTGCTGTCATGTCAACAACCTGGTTAATAACTCAAGAAACTAGCCCATTTTTAAACAACAAAACTATTTTACTATTGACGAGATCTATACCTTCCTTTTAGTGTTAACTTGCTGACTTACAGCACCCACAACTCCTAGCTAGCCAAGTAAACTGTCCAGGAGCCAATTTCACGACAACGAGCCTATATTTTTACCATTATTATACTTCAGTCACAACTCTACTAGCATTTCTGGCAGCCAATATAATTTATTTTTTATTATATTTGAAGCCACAATAATCCCAACCCCCCCTAATTGTAATTACTCGTATGAGGGGCCCAAAAAGAACGCATGTTAGCCGGAAAAACCTACCTAATTTTTTACACCTTATTTGGTCCTGGCCTTCTCACAGCCCTCTGGTACTTTCACCGAGACCTTTGGAACTTGCTCAAGCCACTAGCAAAATTCTTTCCCGGCAAATACCACCCTGTCAACCTGCTCGTTAAACCTGGGTGGCTTGCTTGTCTTATTGCTTCTTGGTCAAAATACCTCTATATGGCGTCCACCTTTGACTACCGAAGGCACATGTCGAATCCCCCATCGCGGGTCTATATTCTTGGCAGGAACCCTCCTCTAACTTGGGGGGCTATGGGATCTTGCGAAATAAAACGCTTTCATTACTGACTCCTTTACACCCCTGGCTCAACCACTTATCGTTTCCTCTCTATTTGGCGTGGCTCCTTTCAGCAATTCTATGTTCCCGTCAAACGGACTCATAAATCCCTAATTGTTCGCATTCTCATCCGTAAGTCACATAGGACTAGATAGAGGATGCGCACGCAAGAGGATTAACTCTACAGAATGAAGCATCACCGGGTCAGTGATAGTGTTATTAATTTCTCACGCCTTGTTCCTCAGGCCATTTGTGCTTGCTAAAACCTCATTCATGAACGAACATCTCCCGCTAAACTTAATTTTACTTCAAAGGACCTCAAATTTATTTTCCCACTGGCTGCAGCGTGATGACTTCTCGCAGCACTCTTATGAATTGGCCCTCATCACCCTCCCCAAAACTTCATTGGGGAAATATCAATTTTAACATCTTATTTCAGTGATCCACATAACACTTACTCTAACCGGGTTAGGCATCATCTTTACAAACTGCCTACTCTTTGTACATATTTTGGGCCTCACAACGAGAGCACCTTCCATCCACTTACACCTACTCCACCCCCACACCCCGGACCCCCTCCTCCTTTCATCCAAACTCCCCCTCCCTCCTTCTACACTGCTATAACCCCCCAACAAACCCTCCTACCTCCACTTATTTAACAACACGCCCCTCCGCCCCCTCCCCCACTCCCACCCTCCTACCCCGTCTCCTCCCCCCCCACTCTCAAAATCCCCCCCCTACCCCTGTCTTGTGATTTACCCCCGAGAAGAGACCCGTCTACATGACTCTCTCCGTTTCACCGAGGCACCTCGACCCGCGCAATGCACTCGAGAAACTGCTAATTACTCGCCACTGAAGTTCAACTCCTCAGCAGGCTCCGCCCCCCCGCCAGAACCTTCCAAAATACATGTACCCCTTTGAGTACCCGCCATTGAGCTTCCCCCCACAAATCTGCCACTAACATACCCCCCCCCATAAATCCTAACTCAGCCCATTACTATCCTCCTCTACTCCTGCCTCCTCCCCCTTCCCTAAACCTCCAAACTCAACAGACCCTTCCCCACCTCAAAAACAAAAACCCGCTGCCAATTACAAACAGGCTTTTCTCATCTCATTAATCCTGCTATTTAATTATTAATGAAAACTCCCAACCCCACCAACAATTTCATGAAAATGATTTAATATTCTGATTACCCCATCAATCTGACCGTTCAGCTTGATCAATACTCTATTCTTTTTATCCAATCGCCCTAAATAGTCTCGTGATGCATTATTGAGTATTCATTATGATACATACACAATGACAACAAATCCAACTCTTTTTCAAATATCTTCATTATCATCTTAACTGGCAATGATACTTTAGTGTCAGCTGGAAAACCTCCTAATGCTATTTATTGGGTGGGAGGGTGGTCGGGATTATTATCATAGCCTCCTCATCGGTGGTACTTCACACGAAAGCAATGCTGGTGCAGCGCGCTTCAAGCGTTCCGTTTTAACCGGAGTGGGAGATATTGGATTTCTGTTCGCCATATTCTGACTAATTTCCTCCTAAGACTCTATTGCCCTAAACTTTATCTTTTTCAATGGAAAGGCCCCACCCCCCTTCTACTAGCCTCATTATTGCAGCCGCCAGCAATTCAGCTCAATTTGGCCTCCCAGCCTTGACTTGGCTTCCGCGATAGAAGGCCGCCACCCCGTATCCGCCCTACTACAACTCTAGCCATAGTTGTTGCGGAGTATTTCCTTCTTCATCGCATCCACCCTTTAATTAAAGATAACCAAACTGCCCTAAACCACCTGCCTATGCTTAGGGGCATTCACTGTGTTTGCTGTACATGTGCTTCTAACCCAAAAATGACGTACAAAAAATTTATGCCTTTTCAACATCAAGTCAGCTTGGTCTGATAGTGGTAGCAATTGGCCTAAACATGCCCCACCTAGCATTTCTTTCATATCTTCACCCACGCTTTTTCAAGGCAATGCTTTTCTTATGGCTCAGGGTCTATCATTCATAGCCTCATCGACGAACTAGGCTATTCGATAAATAGGGGGCTTACAAAAAACTGCCATTTTCAACACAAGTGTGACAATCGGCAGCCTAAGCCCTCATGGGAACCCCCTACTCGCGGGCTTTTTCTCTAAAGACGCATATTATGAGGCAATCAACACCGCCAAACGTAAATGCATGGGCCTACATTAATATCATTGCCACCTCATTCACAGCTGTCTTACCTACGGGTAATTTTCTTTTGCTATCTTTAGACCACCCTCGATTTCCTCCCGCCTCCTCTATTAACGAAAATAACCCACTATATCAGAACCAATTAAACGTCTTGCCGTTGGCAGCATTATTGTGGCCTTTTATTAAATCAGATAATTCCCATCCTCACCAATAAACTTTATAAACAATACCAACCTATTCTTAAAGTTACTGCGATCGCAGTGACTTCCTAGGCTTCCTCATTGCCCTAGACTTAGCTAACGTTTCCTGAACTAAATCGACCGAACAAACAAACCACTCCAAAACAATCAACACCTCTTTTTACCCAACCACCATTCATCGGTCCTCCCACTTATACCATAGACATATGCCTGCGCTTCTCATCTCAATTAATCGGACACCCTCAGGCTAGAAAAAAATTTGGACCAAAAGGCTTAGCAGAACTCCAACTGCCCCATTATAAAAAATCAAGAAATCCAACGAGACAAATTAGAAAACTTACACTCACATCTTTATCTTCACCGCTATTTTATGCTTAATTGTCTTCAGCTCGCTCAAGACCCCACCTTAATGGCCCCGAACTACCTCATATACCGCAAACAGTGTTAATAGTAAAGCTCACAACTGCTATTAATACTCCCCCGCCTCAAAAAAATATTATTGCCACCCGTACCAATCCCCCGCATAAAAACCCCAAACAGCATCCACCACCTCCACTGATTACTGACACCCCATATACTAGAGATGAGAGATATCACCATACCCCAAGCAGCTCATAGATTGTACACAACAATGTCGACCAAAACAAACCCCTCCCCCCCAGGCCTCAGGGTATGGCTCAGCCACAAAGCCGCAGAGTAGGCAAAAACTACTATTTTCCACCTAAGTAAACCAAAAAACAAAACTAAACATAAAAAAGAAGCCCCCCCATCTATCCAAATCTAAAACACCCCGCCCCAGCGGAGCCCACCAACCCCAGAGCAGCAGATAAGGAGAATGGTTTGAAGCCACCGCCAACAGGCCAACAATCAACCCAAGCTCGAATAATACAGTCATAATTCTACAAGGACTTTAAACCTAGACACAGTCCTGAAAAATGTTGTTGTATCAACTATAAGAACTCTAATGGCCCCGTACTTCGCAAAAAACCCAGCCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCTAGTAAAAATTATTAATAAACTCATTTATTGACCTCCCTGCACCCACTAACTTTCGTCATTGTGGAAACTTTGGGGTCTTTCTGGGGTCTGCCTAATTGCCCAAATCGTTACTGGGTTATTCCTAGCGGATACACTTACAACGCTGACACATCTATAGATTCTCATCCGTAGCCCTATTTGCCGAGATGTAAAACAAACGGCTGGCTGCTTCGTAATCTTTCATGCAAACGGCGCCTCATTTTTTCTTCATCTGCATCTACCTCCACATCGGAGAGTGTTTGTACTATGGTTCCTTTCTTATTTAAAGAAACTGAAATATTGGTGTCATTCTCCTATTCCTGGTTCTATGGCTGCAGCATTCGTAGGCTACGTCCTCCCATGAGGACAAATGTCCTTCTGAGGAGCAAACGTAATTACAAACCTTCTCTCCGCTGGCCCCCTATATTGGAAACTTCCGGAAACTGTTCAATGATCTGGGGCGGGTGTTTCAGTAGGACACGCTACTCTGACACGATTTTTCACATTTACTTCATCCTGCCGTTTTCATTGCAGGCGCCTCCATACTTCACCTTCTATTCTTACATCCAAACAGGATCTTCCAATCAACAGGTCTTCACCCCAACTTCGACAGATCCCCTTCCTCGCCTGTTACTCTATAAAGATCTCTTCGGTTCGCAATCATACTTGCCCCTACTTGCCCTACTATCCACTTTTGCCCCCAACCTCCTGGGTGACCCAGACAACTTTACACCAGCCAACCGCTGGTCACCCCCCGCACATCAAGCCAGAGTGATACTTCTTGTCGCTTACGCCATTTCTTCGCTCGATCCCAAATTAAATGGGGGGTCTTGGGCTCTTCTTATTTCTCTATCATAATCCTCTTCCTTCATGCCCTTCCCCATACCTCCAAACAGCGACCCTTATGTTCCGGCCTGGCAAAGCTCTTCCTTTTGAACACTAGTAGGCCAACACCCTAATCCTGACCTGAATCGGAGGTCAGCCAGTAGAAGACCCCTTGTGATAATTGGTCAACTCGCCTCTATTCTCCTACTTGCTTAAATCTTTGTCATCTTGTATCCCCTTACTCGGGACTCACAGAGAACAAATACCCAACTCAGCAGCGACCCACTGGAAGTTACTGTCCAGATGGCACAATTGCTCAACAGACGACCCACTGGAAGTTACTGTCCAAAGACAATTGCTTGCAGTCGACCCACTGGAAGTTACTGTCCAAATGACAATTGCCTTAGTAACTAAATTTCACTCCAATAACCTGCTTATTTAACATGTCATTTAATTCAGTTGCTATACTTCAACAATATTAACCGTCACTAACATGTCTATTTCAATATTATGGTGGCAGTATATGTTCCTGCATTGCGCGGTGTACATATTACTGTATGTATAATAAGACATACTATGTTACTCGCGCATGCAGGACTTTACTGCCCACCATAATATGAAATAGACATGTTAGTGACGGTTTAATATTGTTGAAGTATAGCAGCAACTG 60  PASS    PRECISE;SVTYPE=INS;SVLEN=6543;SUPPORT=47;COVERAGE=298,286,287,282,283;STRAND=+;AC=2;STDEV_LEN=0;STDEV_POS=0;SUPP_VEC=000010000000
----------------------------------------------------------------
DELETION variants with symblic ALT failed like this (this kind of deletions are fine when constructing graph):
[VCFTraversalFinder] Warning: Unable to canonicalize symbolic variant because no reference fasta was given:
NC_058090.1 100355765   Sniffles2.DEL.103C2ME   G   <DEL>   57  PASS    PRECISE;SVTYPE=DEL;SVLEN=-61;END=100355826;SUPPORT=2;COVERAGE=12,12,12,12,13;STRAND=+-;AC=3;STDEV_LEN=64.703;STDEV_POS=377.885;SUPP_VEC=111101110100
----------------------------------------------------------------
same error occured on symblic INVERSION as well:
[VCFTraversalFinder] Warning: Unable to canonicalize symbolic variant because no reference fasta was given:
NC_058080.1_1   1327675 Sniffles2.INV.3EFM0 C   <INV>   60  PASS    PRECISE;SVTYPE=INV;SVLEN=243;END=1327918;SUPPORT=2;COVERAGE=8,4,1,1,6;STRAND=+-;AC=7;STDEV_LEN=284.992;STDEV_POS=8.122;SUPP_VEC=101011111111