Allele mismatch in bcftools step

[averagehat]

Description of the bug

We have successfully run viralrecon with different data sets, but the pipeline fails at the bcftools step in this instance.

Command error:
  Note: applying IUPAC codes based on FORMAT/GT in sample 00284x16
  The fasta sequence does not match the REF allele at DQ181803.1_DENV2:10573:
     REF .vcf: [G]
     ALT .vcf: [A]
     REF .fa : [N]GCTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCGAAACAAAAAACAGCATATTGACGCTGGGAAAGA

Work dir:
  /home/user/user/RUNS/test_viralrecon/30206_UDKV/work/cf/445ad579189aa6e5cdc30408812a24

Container:
  community.wave.seqera.io/library/bcftools_htslib:0a3fa2654b52006f

Command used and terminal output

nextflow run nf-core/viralrecon --skip_bandage -profile docker --input 30206_UKDV_DENV2_ss.csv --outdir ./DENV2_output --platform illumina --protocol metagenomic --fasta 'DQ181803.1_DENV2.fasta' --skip_freyja

Relevant files

Contents of DQ181803.1_DENV2.fasta

>DQ181803.1_DENV2 Dengue virus type 2 strain ThD2_0433_85, complete genome
AGTAGTTAGTCTACGTGGACCGACAAAGACAGATTCTTTGAGGGAGCTAAGCTCAACGTAGTTCTAACAGTTTTTTAATTAGAGAGCAGATCTCTGATGAATAACCAACGGAAAAAGGCGAAAAACACGCCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTGTCGACTGTGCAACAGCTGACAAAGAGATTCTCACTTGGAATGCTGCAGGGGCGAGGACCATTAAAACTGTTCATGGCCCTGGTGGCGTTCCTTCGTTTCCTAACAATCCCACCAACAGCAGGGATATTGAAGAGATGGGGAACAATCAAAAAATCAAAAGCCATTAATGTTTTGAGAGGGTTCAGGAAAGAGATTGGAAGGATGCTGAACATCTTGAATAGGAGACGCAGATCTGCAGGCATGATCATTATGCTGATTCCAACAGTGATGGCGTTCCATCTAACCACACGTAACGGAGAACCACACATGATCGTCAGTAGACAGGAGAAAGGGAAAAGTCTTCTGTTTAAAACAGAGGATGGCGTGAACATGTGCACCCTCATGGCCATGGACCTTGGCGAATTGTGTGAAGACACAATCACGTACAAGTGTCCCCTTCTCAGGCAGAATGAGCCAGAAGACATAGACTGTTGGTGCAACTCCACGTCCACGTGGGTAACCTATGGGACTTGTACCACCACGGGAGAACATAGAAGAGAAAAAAGATCAGTGGCACTCGTTCCACATGTGGGAATGGGACTAGAGACGCGAACTGAAACATGGATGTCATCAGAAGGGGCTTGGAAACATGCCCAGAGAATTGAAACTTGGATCCTGAGACATCCAGGCTTCACCATAATGGCAGCAATCCTGGCATACACCATAGGGACGACACATTTCCAGAGAGCCCTGATTTTCATCCTACTGACAGCTGTCGCTCCTTCAATGACAATGCGTTGCATAGGAATATCAAATAGAGACTTTGTAGAAGGGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGTGTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAAAAACGGAAGCCAAACAGCCTGCCACCCTAAGGAAGTACTGCATAGAAGCAAAGCTAACCAACACAACAACAGAATCTCGTTGCCCAACACAAGGGGAACCCAGCTTAAAAGAAGAGCAGGATAAGAGGTTCGTCTGCAAACACTCCATGGTAGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAGGGAGGCATTGTGACCTGTGCTATGTTCACATGCAAGAAGAACATGGAAGGGAAAATCGTGCAACCAGAAAACTTGGAATACACCATTGTGGTAACACCTCACTCAGGGGAAGAGCATGCGGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAGTAACACCACAGAGTTCCATTACAGAAGCAGAATTGACAGGTTATGGCACCGTCACGATGGAGTGCTCTCCGAGAACAGGCCTCGACTTCAATGAGATGGTGTTGCTGCAGATGGAAAATAAAGCTTGGCTGGTGCATAGGCAATGGTTCCTAGACCTGCCATTACCATGGCTGCCCGGAGCGGATACACAAGGGTCAAATTGGATACAGAAAGAAACATTGGTCACTTTCAAAAATCCCCATGCGAAGAAACAGGATGTTGTTGTTTTAGGATCCCAAGAAGGGGCCATGCATACAGCACTCACAGGAGCCACAGAAATCCAAATGTCATCAGGAAACTTACTCTTCACTGGACATCTCAAGTGCAGGCTGAGAATGGACAAGCTACAGCTCAAAGGAATGTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAGAAACACAACATGGAACGATAGTTATCAGAGTGCAATATGAAGGGGACGGCTCTCCATGTAAAATCCCTTTTGAGATAATGGATTTGGAAAAAAGATATGTCTTAGGCCGCCTGATCACAGTCAACCCAATTGTGACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATAGGAGTAGAGCCGGGACAACTGAAGCTCAACTGGTTTAAGAAAGGAAGTTCTATCGGCCGAATGTTTGAGACAACAATGAGGGGGGCGAAGAGAATGGCCATTTTGGGTGACACAGCCTGGGATTTCGGATCCCTGGGAGGAGTGTTTACATCTATAGGAAAAGCTCTCCACCAAGTCTTTGGAGCAATCTATGGAGCTGCCTTCAGTGGGGTTTCATGGACTATGAAAATCCTCATAGGAGTCATTATCACATGGATAGGAATGAATTCACGCAGCACCTCACTGTCTGTGTCACTAGTATTGGTGGGAATTGTGACACTGTATTTGGGAGTCATGGTGCAGGCCGATAGTGGTTGCGTTGTGAGCTGGAAAAACAAAGAACTGAAATGTGGCAGTGGGATTTTCATCACAGACAACGTGCACACATGGACAGAACAATACAAGTTCCAACCAGAATCCCCTTCAAAACTAGCTTCAGCTATCCAGAAAGCTCAAGAAGAGGGAATTTGTGGAATCCGCTCAGTAACAAGATTGGAGAACCTAATGTGGAAACAAATAACACCAGAATTGAATCACATTCTAGCAGAAAATGAGGTGAAGTTAACTATCATGACAGGAGACATCAAAGGAATCATGCAGGCAGGAAAACGATCTCTGCGGCCTCAGCCCACTGAGCTAAAGTATTCATGGAAAACATGGGGCAAAGCAAAAATGATCTCCACAGAGTCTCATAACCAAACCTTTCTCATTGATGGCCCCGAAACAGCAGAATGCCCCAACACAAATAGAGCTTGGAACTCGTTGGAGGTTGAAGACTACGGCTTTGGAGTGTTCACCACCAATATATGGCTAAAATTGAAAGAAAAACAGGATGCATTCTGCGACTCAAAACTCATGTCAGCGGCCATAAAAGACAACAGAGCCGTCCATGCCGACATGGGTTATTGGATAGAAAGTGCACTCAATGACACATGGAAGATAGAGAAAGCCTCTTTCATTGAAGTAAAAAACTGCCACTGGCCAAAATCACACACCCTCTGGAGCAATGGAGTGCTAGAAAGTGAGATGATAATTCCAAAGAATCTCGCTGGACCAGTGTCTCAACACAACTATAGACCAGGCTACCATACACAAATAGCAGGACCATGGCATCTAGGTAAGCTTGAGATGGACTTTGATTTCTGCGATGGAACTACAGTGGTAGTGACTGAGGACTGCGGAAATAGAGGACCCTCTTTGAGAACAACCACTGCCTCTGGAAAACTCATAACAGAATGGTGCTGCCGATCTTGCACATTACCACCCCTAAGATACAGAGGTGAGGATGGGTGCTGGTACGGGATGGAAATCAGACCATTGAAAGAGAAAGAAGAAAATTTGGTTAACTCCTTGGTCACAGCCGGACATGGGCAGGTTGACAATTTTTCACTAGGAGTCTTGGGAATGGCATTGTTCCTGGAGGAAATGCTTAGGACCCGAGTAGGAACGAAACATGCAATACTACTAGTTGCAGTTTCTTTTGTGACATTGATCACAGGGAACATGTCCTTTAAAGACCTAGGAAGAGTGGTGGTTATGGTAGGCGCCACCATGACGGATGACATAGGTATGGGCGTGACTTATCTTGCCCTACTAGCAGCCTTCAAAGTTAGACCAACTTTTGCAGCTGGACTACTCTTGAGAAAGCTGACCTCCAAGGAATTGATGATGACTACCATAGGAATTGTACTCCTCTCCCAAAGCACTATACCAGAGACCATTCTTGAATTGACTGATGCATTAGCCTTAGGCATGATGGTCCTCAAAATGGTAAGAAACATGGAGAAGTATCAGTTAGCAGTGACTATCATGGCTATCTTGTGCGTCCCAAACGCAGTGATATTACAAAATGCATGGAAAGTGAGTTGCACAATACTGGCAGTGGTGTCCGTTTCCCCACTGCTTTTGACATCCTCACAGCAAAAAACGGATTGGATACCATTAGCATTGACAATCAAGGGTCTCAATCCAACAGCCATTTTTCTAACAACCCTCTCAAGAACCAGCAAGAAAAGGAGCTGGCCACTAAATGAGGCTATCATGGCAGTCGGAATGGTGAGCATTTTAGCCAGTTCTCTCCTAAAAAATGATATTCCCATGACAGGACCACTAGTGGCTGGAGGGCTCCTCACTGTGTGCTACGTGCTCACTGGACGATCGGCCGACTTGGAACTGGAGAGAGCAACCGATGTCAAATGGGAAGACCAGGCAGAGATATCAGGAAGCAGTCCAATCCTGTCAATAACAATATCAGAAGATGGTAGCATGTCGATAAAAAATGAAGAGGAAGAACAAACACTGACCATACTCATTAGAACAGGATTGCTGGTGATCTCAGGACTTTTTCCTGTATCAATACCAATCACGGCAGCAGCATGGTACCTGTGGGAAGTGAAGAAACAACGGGCCGGAGTATTGTGGGATGTTCCTTCACCCCCACCCATGGGAAAGGCTGAACTGGAAGATGGAGCCTACAGAATTAAGCAAAAAGGGATTCTTGGATATTCCCAGATCGGAGCTGGAGTTTATAAAGAAGGAACATTCCATACAATGTGGCATGTCACACGTGGCGCTGTCCTAATGCACAAAGGAAAGAGGATTGAACCATCATGGGCGGACGTCAAAAAAGATCTAATATCATATGGAGGAGGCTGGAAGTTAGAAGGAGAATGGAAGGAAGGAGAAGAAGTCCAGGTGCTGGCATTGGAGCCTGGAAAAAATCCAAGAGCCGTCCAAACAAAACCTGGTCTTTTCAAAACCAACGCCGGAACAATAGGTGCCGTATCTCTGGACTTTTCCCCTGGAACGTCAGGATCTCCAATCATTGACAAAAAAGGAAAAGTTGTGGGTCTTTATGGTAATGGTGTTGTTACAAGGAGTGGAGCATATGTGAGTGCTATAGCCCAAACTGAAAAAAGCATTGAAGACAACCCAGAGATCGAAGATGACATTTTCCGAAAGAGAAGACTGACCATCATGGACCTCCACCCAGGAGCGGGAAAGACGAAAAGATACCTTCCGGCCATAGTCAGAGAAGCTATAAAACGGGGTTTGAGAACATTAATCTTGGCCCCCACCAGAGTTGTGGCAGCTGAAATGGAGGAAGCTCTCAGAGGACTTCCAATAAGATACCAAACCCCAGCCATCAGAGCTGAGCACACTGGGCGGGAAATTGTGGATCTAATGTGTCATGCCACATTTACCATGAGGCTGCTATCACCAGTTAGAGTGCCAAACTACAACCTGATTATCATGGACGAAGCCCATTTCACAGACCCAGCAAGTATAGCAGCTAGAGGATACATCTCAACTCGAGTGGAGATGGGTGAGGCAGCTGGGATTTTCATGACAGCCACTCCCCCGGGAAGCAGAGATCCATTTCCTCAGAGCAATGCACCAATCATAGATGAAGAAAGAGAAATCCCTGAACGTTCATGGAATTCTGGACATGAGTGGGTCACGGATTTTAAAGGGAAGACTGTTTGGTTCGTTCCAAGCATAAAAGCAGGAAATGATATAGCAGCTTGCCTGAGAAAAAATGGAAAGAAAGTAATACAACTTAGTAGGAAGACTTTTGATTCTGAGTATGTCAAGACTAGAACCAACGATTGGGATTTCGTGGTTACAACTGACATTTCAGAAATGGGTGCCAATTTCAAGGCTGAGAGGGTTATAGACCCCAGACGCTGCATGAAACCAGTCATACTAACAGATGGTGAGGAGCGGGTGATTCTGGCAGGACCTATGCCAGTGACCCACTCTAGTGCAGCACAAAGAAGAGGGAGAATAGGAAGAAATCCAAAAAATGAAAATGACCAGTACATATACATGGGGGAACCTCTGGAAAATGATGAAGACTGTGCACACTGGAAAGAAGCCAAAATGCTCCTAGATAACATTAACACACCAGAAGGAATCATTCCCAGTATGTTTGAACCAGAGCGTGAAAAAGTGGATGCCATTGATGGCGAATACCGCTTGAGAGGAGAAGCAAGGAAAACCTTTGTGGACTTAATGAGAAGAGGAGACCTACCAGTCTGGTTGGCCTACCAAGTGGCAGCTGAAGGCATCAACTATGCAGACAGAAGGTGGTGTTTTGATGGAATCAAGAACAACCAAATCCTGGAAGAAAATGTGGAAGTTGAAATCTGGACAAAAGAAGGGGAAAGGAAGAAATTGAAACCCAGATGGCTGGATGCTAGGATCTATTCTGACCCACTGGCGCTAAAAGAATTCAAAGAATTTGCAGCCGGAAGAAAGTCTCTGACCCTGAACCTAATCACAGAAATGGGTAGGCTTCCAACATTCATGACTCAGAAGACAAGAGACGCACTGGACAACTTAGCAGTGCTGCACACGGCTGAAGCAGGTGGAAGGGCGTACAATCATGCTCTCAGTGAACTGCCGGAGACCCTGGAGACACTGCTTTTACTGACACTTCTGGCTACAGTCACGGGAGGGATCTTTTTATTCTTGATGAGCGGGAGGGGCATAGGGAAGATGACCCTGGGAATGTGCTGCATAATCACGGCTAGCATCCTTCTATGGTACGCACAAATACAGCCACATTGGATAGCAGCTTCAATAATACTGGAGTTTTTTCTCATAGTTTTGCTTATTCCAGAACCAGAAAAACAGAGAACACCCCAAGACAACCAATTGACCTACGTTGTCATAGCCATCCTCACAGTGGTGGCCGCAACCATGGCAAACGAGATGGGTTTCCTAGAAAAAACGAAGAAAGATCTTGGATTAGGAAGCATTGCAACCCAGCAACCCGAGAGCAACATCCTGGACATAGATCTACGTCCCGCATCAGCATGGACGCTGTATGCCGTGGCCACAACATTTGTTACACCAATGCTGAGACATAGCATTGAGAATTCCTCAGTGAATGTATCACTAACAGCTATAGCCAACCAAGCAACAGTGTTAATGGGTCTAGGGAAAGGATGGCCATTGTCAAAGATGGACATCGGAGTTCCCCTTCTCGCCATTGGATGCTACTCACAAGTCAATCCCATAACTCTCACAGCAGCTCTTCTCTTATTGGTAGCGCATTATGCCATCATAGGGCCAGGACTTCAAGCAAAAGCAACCAGAGAAGCTCAGAAAAGAGCAGCGGCGGGCATCATGAAAAACCCAACCGTCGATGGAATAACAGTGATTGATCTAGATCCAATACCTTATGATCCAAAGTTTGAAAAGCAGTTGGGACAAGTAATGCTCCTAGTCCTCTGCGTGACTCAAGTATTGATGATGAGGACTACATGGGCTCTGTGTGAGGCCTTAACCTTAGCTACCGGGCCCATCTCCACACTGTGGGAAGGAAATCCAGGAAGGTTCTGGAACACTACCATTGCGGTGTCAATGGCTAACATCTTTAGAGGGAGTTACTTGGCTGGAGCTGGACTTCTCTTTTCCATTATGAAGAACACAACCAACACAAGAAGGGGAACTGGCAACATAGGAGAGACGCTTGGAGAGAAATGGAAAAGCCGATTGAACGCACTGGGAAAAAGTGAATTCCAGATCTACAAGAAAAGTGGAATCCAGGAAGTGGATAGAACCTTAGCAAAAGAAGGCATCAAAAGAGGAGAAACGGACCATCACGCTGTGTCGCGAGGCTCAGCAAAACTGAGATGGTTCGTTGAGAGAAACATGGTCACACCAGAAGGGAAAGTAGTGGACCTCGGTTGTGGCAGAGGAGGCTGGTCATACTATTGTGGAGGACTAAAAAACGTAAGAGAAGTGAAAGGCCTAACAAAAGGAGGACCAGGACACGAAGAACCCATCCCCATGTCAACATATGGGTGGAATCTAGTGCGCCTTCAAAGTGGAGTTGACGTTTTCTTCTTCCCGCCAGAAAAGTGTGACACATTGTTGTGTGACATAGGGGAGTCATCACCAAATCCCACAGTGGAAGCAGGACGAACACTCAGAGTCCTTAATTTAGTAGAAAATTGGTTGAACAACAACACTCAATTTTGCATAAAAGTTCTCAATCCATATATGCCCTCAGTTATAGAAAAAATGGAAACACTGCAAAGGAAATATGGAGGAGCCTTAGTGAGAAATCCACTCTCACGGAACTCCACACATGAGATGTACTGGGTATCCAATGCTTCCGGGAACATAGTGTCATCAGTGAACATGATTTCAAGGATGTTGATCAACAGATTCACAATGAGACACAAGAAAGCCACTTACGAGCCGGATGTTGACCTCGGAAGCGGAACCCGTAACATTGGAATTGAAAGTGAGATACCAAACCTAGACATAATTGGGAAAAGAATAGAAAAAATAAAGCAAGAGCATGAAACATCATGGCACTACGACCAAGACCACCCATACAAAACGTGGGCATACCATGGTAGCTATGAAACAAAACAGACTGGATCAGCATCATCCATGGTCAACGGAGTGGTCAGGCTGCTGACAAAACCCTGGGACGTCCTCCCCACGGTGACACAGATGGCAATGACAGACACGACTCCATTTGGACAACAGCGTGTTTTTAAAGAAAAAGTGGACACGAGAACCCAAGAACCGAAAGAAGGCACGAAGAAACTAATGAAAATCACAGCAGAGTGGCTGTGGAAAGAATTAGGGAAGAAAAAGACACCTAGGATGTGCACCAGAGAAGAATTCACAAGAAAGGTGAGAAGCAATGCAGCCTTGGGGGCCATATTCACTGATGAGAACAAGTGGAAGTCGGCACGTGAGGCTGTTGAAGATAGTAGGTTTTGGGAGCTGGTTGACAAGGAAAGGAATCTCCATCTTGATGGAAAGTGTGAAACATGTGTGTACAACATGATGGGAAAAAGAGAAAAGAAGCTAGGGGAATTCGGCAAGGCAAAAGGCAGCAGAGCCATATGGTACATGTGGCTTGGAGCTCGCTTCCTGGAGTTTGAAGCCCTAGGATTCCTAAATGAAGATCACTGGTTCTCCAGAGAGAACTCCCTGAGTGGAGTGGAAGGAGAAGGGCTGCACAAGCTAGGTTACATTCTAAGAGACGTGAGCAAGAAAGAGGGAGGAGCCATGTATGCCGATGACACAGCAGGATGGGACACAAGAATCACACTAGAAGACCTAAAAAATGAAGAAATGGTAACAAACCACATGGAAGGAGAACACAAGAAATTAGCCGAGGCCATTTTCAAACTAACGTACCAAAACAAGGTGGTGCGTGTGCAAAGACCAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGAGGTAGTGGGCAAGTTGGTACCTATGGACTCAACACTTTCACCAATATGGAAGCCCAATTAATCAGACAGATGGAGGGAGAAGGAGTCTTTAAAAACATTCAGCACCTGACAGTCACAGAAGAAACCGCCGTGCAAAACTGGTTAGCAAGAGTGGGGCGCGAAAGGTTATCAAGAATGGCCATCAGTGGAGATGATTGTGTTGTGAAACCTTTAGATGACAGATTCGCAAGCGCTTTGACAGCTCTAAATGACATGGGAAAGATTAGAAAAGACATACAACAATGGGAACCTTCAAGAGGATGGAACGATTGGACACAAGTGCCCTTCTGTTCACACCATTTCCATGAGCTAATCATGAAGGATGGTCGTGTACTTGTTGTTCCATGTAGAAACCAAGATGAACTGATTGGCAGAGCCCGAATCTCCCAAGGAGCAGGGTGGTCTTTGCGGGAGACGGCCTGTTTGGGGAAGTCTTACGCCCAAATGTGGAGCTTGATGTATTTCCACAGACGCGACCTCAGGCTGGCGGCAAATGCCATTTGCTCGGCAGTGCCATCACATTGGGTTCCAACAAGTCGAACAACCTGGTCCATACACGCCAAACATGAATGGATGACAACGGAAGACATGCTGACAGTCTGGAACAGGGTGTGGATTCAAGAAAACCCATGGATGGAAGACAAAACCCCAGTGGAATCATGGGAGGAAATCCCATACTTGGGGAAAAGAGAAGACCAATGGTGCGGCTCATTGATTGGGTTAACAAGCAGGGCCACATGGGCAAAGAACATCCAAGCAGCAATAAATCAAGTTAGATCTCTTATAGGCAATGAGGAATACACAGACTACATGCCATCCATGAAAAGATTCAGAAGAGAAGAGGAAGAAGCAGGAGTCCTGTGGTAGAAAGCAGAACTAACATAAAACAAGGCTAGAAGTCAGGTCGGATTAAGCCATAGTACGGAAAAAACTATGCTACCTGTGAGCCCCGTCCAAGGACGTTAAAAGAAGTCAGGCCATCATAAATGCCATAGCTTGAGTAAACTATGCAGCCTGTAGCTCCACCTGAGAAGGTGTAAAAAATCTGGGAGGCCACAAACCATGGAAGCTGTACGCATGGCGTAGTGGACTAGCGGTTAGAGGAGACCCCTCCCTTACAAATCGCAGCAACAATGGGGGCCCAAGGCGAGATGAAGCTGTAGTCTCGCTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCGAAACAAAAAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAGAACGCCAGAAAATGGAATGGTGCTGTTGAATCAACAGGTTCT

System information

N E X T F L O W ~ version 25.04.6
Rocky Linux release 9.7
nf-core/viralrecon 3.0.0

[svarona]

Can you send us the information of the variant DQ181803.1_DENV2:10573:G >A in that position? Total depth, alternative allele depth, allele frequency...

We used to have that problem but it happened when the depth thresholds between consensus masking and variant calling are not the same. I assume you are using default ones, right?

Can you confirm which variant caller and consensus caller you are using?

[averagehat]

Can you send us the information of the variant DQ181803.1_DENV2:10573:G >A in that position? Total depth, alternative allele depth, allele frequency...

DQ181803.1_DENV2        10573   .       G       A       228.302 PASS    DP=176;ADF=13,135;ADR=0,28;AD=13,163;VDB=0.186323;SGB=-0.693147;RPBZ=1.27568;MQBZ=6.84662;MQSBZ=-3.62318;BQBZ=-0.491965;SCBZ=-2.35675;MQ0F=0;AC=1;AN=1;DP4=13,0,135,28;MQ=41    GT:PL:DP:SP:ADF:ADR:AD  1:255,0:176:6:13,135:0,28:13,163

We used to have that problem but it happened when the depth thresholds between consensus masking and variant calling are not the same. I assume you are using default ones, right?

We are using the defaults, unless the command I posted somehow changes those.

Can you confirm which variant caller and consensus caller you are using?

The default is BCF tools, I think? I think this should be clear from my original post, which has the full options selected.

[svarona]

I think that unless you specify the viralrecon version when running nextflow -r 3.0.0 it automatically uses the version 2.6.0. Can you double check that too please?

The default consensus caller is bcftools, and the default variant caller depends on the protocol

viralrecon/conf/modules_illumina.config

Line 14 in 395079f

if (!variant_caller) { variant_caller = params.protocol == 'amplicon' ? 'ivar' : 'bcftools' }

So for metagenomic is bcftools.

Is it possible that you are having a deletion or something close to that position?

Have you opened the .bam files in IGV to see what is going on there?