Variant not detected within 16bp from the start/end

[peterk87]

Hello,

I noticed that Clair3 is not calling a variant near the end of a short reference sequence (position 2265 of Influenza A virus PB2 sequence OP597571.1; sequence length=2280).

Bcftools calls at a G->A variant at position 2265. It's also clear from looking at the read alignment in IGV that there's a high AF variant at the position.

Clair3 merge_output.vcf.gz:

##fileformat=VCFv4.2
##source=Clair3
##clair3_version=1.0.4
##cmdline=../run_clair3.sh --bam_fn=minimap2.bam --ref_fn=OP597571.1.fasta --model_path=.../models/r941_prom_sup_g5014 --print_ref_calls --gvcf --threads=1 --platform=ont --output=clair3-out --haploid_sensitive --enable_long_indel --keep_iupac_bases --no_phasing_for_fa --include_all_ctgs
##reference=OP597571.1.fasta
##FILTER=<ID=PASS,Description="All filters passed">
##FILTER=<ID=LowQual,Description="Low quality variant">
##FILTER=<ID=RefCall,Description="Reference call">
##INFO=<ID=P,Number=0,Type=Flag,Description="Result from pileup calling">
##INFO=<ID=F,Number=0,Type=Flag,Description="Result from full-alignment calling">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ<20 or selected by 'samtools view -F 2316' are filtered)">
##FORMAT=<ID=AD,Number=R,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
##FORMAT=<ID=AF,Number=1,Type=Float,Description="Observed allele frequency in reads, for each ALT allele, in the same order as listed, or the REF allele for a RefCall">
##contig=<ID=OP597571.1,length=2280>
#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  SAMPLE
OP597571.1      62      .       C       .       17.62   RefCall P       GT:GQ:DP:AD:AF:PL       0:17:828:641:0.7742:990
OP597571.1      185     .       G       A       9.65    PASS    F       GT:GQ:DP:AD:AF:PL       1:9:143:2,107:0.7483:18,13,0
OP597571.1      232     .       T       .       21.36   RefCall P       GT:GQ:DP:AD:AF:PL       0:21:102:75:0.7353:990
OP597571.1      252     .       C       A       25.12   PASS    P       GT:GQ:DP:AD:AF:PL       1:25:90:1,84:0.9333:80,58,0
OP597571.1      495     .       G       .       19.81   RefCall P       GT:GQ:DP:AD:AF:PL       0:19:42:36:0.8571:990
OP597571.1      510     .       C       T       33.42   PASS    F       GT:GQ:DP:AD:AF:PL       1:33:42:0,41:0.9762:79,57,0
OP597571.1      528     .       A       C       35.38   PASS    F       GT:GQ:DP:AD:AF:PL       1:35:44:0,41:0.9318:80,63,0
OP597571.1      540     .       G       C       26.34   PASS    P       GT:GQ:DP:AD:AF:PL       1:26:45:0,44:0.9778:80,54,0
OP597571.1      639     .       C       .       22.44   RefCall P       GT:GQ:DP:AD:AF:PL       0:22:44:28:0.6364:990
OP597571.1      694     .       T       .       26.30   RefCall P       GT:GQ:DP:AD:AF:PL       0:26:43:37:0.8605:990
OP597571.1      708     .       A       .       23.57   RefCall P       GT:GQ:DP:AD:AF:PL       0:23:43:33:0.7674:990
OP597571.1      741     .       A       .       23.27   RefCall P       GT:GQ:DP:AD:AF:PL       0:23:49:30:0.6122:990
OP597571.1      747     .       A       G       24.68   PASS    F       GT:GQ:DP:AD:AF:PL       1:24:47:4,40:0.8511:74,37,0
OP597571.1      888     .       T       C       24.92   PASS    P       GT:GQ:DP:AD:AF:PL       1:24:58:1,56:0.9655:80,42,0
OP597571.1      895     .       A       .       23.47   RefCall P       GT:GQ:DP:AD:AF:PL       0:23:59:51:0.8644:990
OP597571.1      1113    .       A       .       21.30   RefCall P       GT:GQ:DP:AD:AF:PL       0:21:45:40:0.8889:990
OP597571.1      1170    .       C       T       17.59   PASS    F       GT:GQ:DP:AD:AF:PL       1:17:41:25,12:0.2927:22,0,63
OP597571.1      1335    .       G       A       28.63   PASS    F       GT:GQ:DP:AD:AF:PL       1:28:43:0,40:0.9302:77,45,0
OP597571.1      1345    .       T       .       25.67   RefCall P       GT:GQ:DP:AD:AF:PL       0:25:44:30:0.6818:990
OP597571.1      1432    .       G       .       24.87   RefCall P       GT:GQ:DP:AD:AF:PL       0:24:52:43:0.8269:990
OP597571.1      1469    .       A       G       24.83   PASS    P       GT:GQ:DP:AD:AF:PL       1:24:52:2,49:0.9423:80,37,0
OP597571.1      1504    .       T       .       22.74   RefCall P       GT:GQ:DP:AD:AF:PL       0:22:50:41:0.8200:990
OP597571.1      1522    .       A       .       26.06   RefCall P       GT:GQ:DP:AD:AF:PL       0:26:54:39:0.7222:990
OP597571.1      1566    .       G       A       32.47   PASS    F       GT:GQ:DP:AD:AF:PL       1:32:54:2,52:0.9630:80,53,0
OP597571.1      1677    .       T       C       31.74   PASS    F       GT:GQ:DP:AD:AF:PL       1:31:57:4,49:0.8596:80,52,0
OP597571.1      1736    .       C       .       23.60   RefCall P       GT:GQ:DP:AD:AF:PL       0:23:59:41:0.6949:990
OP597571.1      1737    .       T       .       25.37   RefCall P       GT:GQ:DP:AD:AF:PL       0:25:59:43:0.7288:990
OP597571.1      1760    .       C       .       21.45   RefCall P       GT:GQ:DP:AD:AF:PL       0:21:60:52:0.8667:990
OP597571.1      1761    .       T       .       18.29   RefCall P       GT:GQ:DP:AD:AF:PL       0:18:60:44:0.7333:990
OP597571.1      1806    .       G       A       27.10   PASS    P       GT:GQ:DP:AD:AF:PL       1:27:58:0,54:0.9310:80,47,0
OP597571.1      1942    .       C       .       21.32   RefCall P       GT:GQ:DP:AD:AF:PL       0:21:87:73:0.8391:990
OP597571.1      1957    .       T       .       24.29   RefCall P       GT:GQ:DP:AD:AF:PL       0:24:96:72:0.7500:990
OP597571.1      2051    .       T       C       26.04   PASS    P       GT:GQ:DP:AD:AF:PL       1:26:168:1,156:0.9286:80,41,0
OP597571.1      2154    .       A       .       32.13   RefCall F       GT:GQ:DP:AD:AF:PL       0:32:713:468:0.6564:990
OP597571.1      2216    .       G       .       21.01   RefCall P       GT:GQ:DP:AD:AF:PL       0:21:834:715:0.8573:990
OP597571.1      2253    .       C       .       18.05   RefCall P       GT:GQ:DP:AD:AF:PL       0:18:767:569:0.7419:990

Bcftools mpileup and call output:

##fileformat=VCFv4.2
##FILTER=<ID=PASS,Description="All filters passed">
##bcftoolsVersion=1.17+htslib-1.17
##bcftoolsCommand=bcftools mpileup -d 100000 -f OP597571.1.fasta minimap2.bam
##contig=<ID=OP597571.1,length=2280>
##ALT=<ID=*,Description="Represents allele(s) other than observed.">
##INFO=<ID=INDEL,Number=0,Type=Flag,Description="Indicates that the variant is an INDEL.">
##INFO=<ID=IDV,Number=1,Type=Integer,Description="Maximum number of raw reads supporting an indel">
##INFO=<ID=IMF,Number=1,Type=Float,Description="Maximum fraction of raw reads supporting an indel">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">
##INFO=<ID=VDB,Number=1,Type=Float,Description="Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is better)",Version="3">
##INFO=<ID=RPBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Read Position Bias (closer to 0 is better)">
##INFO=<ID=MQBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Mapping Quality Bias (closer to 0 is better)">
##INFO=<ID=BQBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Base Quality Bias (closer to 0 is better)">
##INFO=<ID=MQSBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Mapping Quality vs Strand Bias (closer to 0 is better)">
##INFO=<ID=SCBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Soft-Clip Length Bias (closer to 0 is better)">
##INFO=<ID=SGB,Number=1,Type=Float,Description="Segregation based metric, http://samtools.github.io/bcftools/rd-SegBias.pdf">
##INFO=<ID=MQ0F,Number=1,Type=Float,Description="Fraction of MQ0 reads (smaller is better)">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="List of Phred-scaled genotype likelihoods">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes for each ALT allele, in the same order as listed">
##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">
##INFO=<ID=DP4,Number=4,Type=Integer,Description="Number of high-quality ref-forward , ref-reverse, alt-forward and alt-reverse bases">
##INFO=<ID=MQ,Number=1,Type=Integer,Description="Average mapping quality">
##bcftools_callVersion=1.17+htslib-1.17
##bcftools_callCommand=call --ploidy 1 -mv -Ob -o calls.vcf; Date=Mon Dec 18 20:32:30 2023
#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  WIN-AH-2023-FAV-0375-3-1C2d.Segment_1_PB2.OP597571.1.bam
OP597571.1      185     .       G       A       228.391 .       DP=139;VDB=0.979122;SGB=-0.693147;RPBZ=-0.747496;MQBZ=-0.574548;MQSBZ=-0.192448;BQBZ=1.13137;SCBZ=-0.508121;MQ0F=0;AC=1;AN=1;DP4=1,1,29,40;MQ=56       GT:PL   1:255,0
OP597571.1      252     .       C       A       225.421 .       DP=102;VDB=0.747331;SGB=-0.693147;MQSBZ=-0.642482;MQ0F=0;AC=1;AN=1;DP4=0,0,27,46;MQ=55  GT:PL 1:255,0
OP597571.1      510     .       C       T       228.402 .       DP=42;VDB=0.0719096;SGB=-0.693145;RPBZ=-0.412677;MQBZ=0;MQSBZ=0;BQBZ=1.36377;SCBZ=0.323986;MQ0F=0;AC=1;AN=1;DP4=1,0,16,25;MQ=60        GT:PL   1:255,0
OP597571.1      528     .       A       C       211.417 .       DP=41;VDB=0.278679;SGB=-0.693145;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,19,22;MQ=60   GT:PL   1:241,0
OP597571.1      540     .       G       C       225.417 .       DP=44;VDB=0.219325;SGB=-0.693146;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,18,26;MQ=60   GT:PL   1:255,0
OP597571.1      741     .       AGGGG   AGG     17.0828 .       INDEL;IDV=18;IMF=0.367347;DP=49;VDB=0.73002;SGB=-0.683931;RPBZ=-0.778151;MQBZ=0.124761;MQSBZ=-0.650785;BQBZ=1.02751;SCBZ=0.358316;MQ0F=0;AC=1;AN=1;DP4=14,17,8,5;MQ=58 GT:PL   1:87,42
OP597571.1      747     .       A       G       149.305 .       DP=45;VDB=0.841016;SGB=-0.693145;RPBZ=1.48153;MQBZ=1.82034;MQSBZ=0;BQBZ=2.48105;SCBZ=0.918559;MQ0F=0;AC=1;AN=1;DP4=4,1,19,21;MQ=58     GT:PL   1:176,0
OP597571.1      888     .       T       C       228.415 .       DP=57;VDB=0.0352829;SGB=-0.693147;RPBZ=0.304058;MQBZ=-0.190662;MQSBZ=0.201778;BQBZ=1.7129;SCBZ=0.309711;MQ0F=0;AC=1;AN=1;DP4=1,0,24,32;MQ=58   GT:PL   1:255,0
OP597571.1      1335    .       G       A       228.424 .       DP=42;VDB=0.713753;SGB=-0.693145;RPBZ=0.0412661;MQBZ=-0.156174;MQSBZ=-0.866025;BQBZ=1.69535;SCBZ=0.481876;MQ0F=0;AC=1;AN=1;DP4=1,0,17,24;MQ=59 GT:PL   1:255,0
OP597571.1      1469    .       A       G       221.38  .       DP=52;VDB=0.389159;SGB=-0.693147;RPBZ=0.667379;MQBZ=-0.353341;MQSBZ=-1.08356;BQBZ=2.5377;SCBZ=-1.22412;MQ0F=0;AC=1;AN=1;DP4=2,1,17,32;MQ=59    GT:PL   1:248,0
OP597571.1      1566    .       G       A       228.393 .       DP=55;VDB=0.00601305;SGB=-0.693147;RPBZ=0.899577;MQBZ=-0.498471;MQSBZ=-1.09919;BQBZ=1.93931;SCBZ=-1.1883;MQ0F=0;AC=1;AN=1;DP4=1,1,19,34;MQ=56  GT:PL   1:255,0
OP597571.1      1677    .       T       C       203.339 .       DP=55;VDB=0.0325114;SGB=-0.693147;RPBZ=-0.145875;MQBZ=-0.280056;MQSBZ=-0.785905;BQBZ=1.93259;SCBZ=-0.687735;MQ0F=0;AC=1;AN=1;DP4=2,2,19,32;MQ=58       GT:PL   1:230,0
OP597571.1      1806    .       G       A       228.42  .       DP=55;VDB=0.0163287;SGB=-0.693147;RPBZ=0.346711;MQBZ=-0.136083;MQSBZ=-0.847791;BQBZ=1.67811;SCBZ=-1.38535;MQ0F=0;AC=1;AN=1;DP4=1,0,22,32;MQ=59 GT:PL   1:255,0
OP597571.1      2051    .       T       C       228.414 .       DP=185;VDB=0.258521;SGB=-0.693147;RPBZ=-0.659562;MQBZ=-0.811578;MQSBZ=-6.21909;BQBZ=2.58014;SCBZ=-0.128242;MQ0F=0;AC=1;AN=1;DP4=1,2,100,80;MQ=55       GT:PL   1:255,0
OP597571.1      2265    .       G       A       228.4   .       DP=802;VDB=0;SGB=-0.693147;RPBZ=-0.477099;MQBZ=2.06864;MQSBZ=3.44984;BQBZ=7.12028;SCBZ=0.475257;MQ0F=0;AC=1;AN=1;DP4=23,0,710,60;MQ=54 GT:PL   1:255,0

Clair3 command:

CLAIR_BIN_DIR=$(dirname $(which run_clair3.sh))
MODEL_PATH="$CLAIR_BIN_DIR/models/r941_prom_sup_g5014"

samtools faidx ref.fasta

run_clair3.sh \
    --bam_fn=minimap2.bam \
    --ref_fn=OP597571.1.fasta \
    --model_path="$MODEL_PATH"\
    --threads=1 \
    --platform="ont" \
    --output=clair3-out \
    --haploid_sensitive \
    --enable_long_indel \
    --keep_iupac_bases \
    --no_phasing_for_fa \
    --include_all_ctgs

Other details:

• Clair3 version: 1.0.4 (installed from Bioconda)
• read mapper: Minimap2 v2.24
• instrument: GridION
• instrument software: MinKNOW 23.07.12 (Bream 7.7.6, Core 5.7.5, Dorado 7.1.4+d7df870c0)
• basecalling model: Super-accurate basecalling, 450 bps
• flowcell: FLO-MIN106
• kit: SQK-RBK110-96

---

Are there any parameters that need to be adjusted for variant calling of RNA virus sequence data?

Any help would be much appreciated!

Thanks!

[aquaskyline]

Could you please try Clair3 with the --vcf_fn= option with the VCF called by bcftools. That forces Clair3 to do genotyping at site 2265. Please show the result at 2265 if it runs successfully.

[peterk87]

Sure, here's the VCF output for each vcf.gz in the output dir:

merge_output.vcf.gz:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  SAMPLE
OP597571.1      185     .       G       A       9.65    PASS    F       GT:GQ:DP:AD:AF:PL       1:9:143:2,107:0.7483:18,13,0
OP597571.1      252     .       C       A       25.12   PASS    P       GT:GQ:DP:AD:AF:PL       1:25:90:1,84:0.9333:80,58,0
OP597571.1      510     .       C       T       33.42   PASS    F       GT:GQ:DP:AD:AF:PL       1:33:42:0,41:0.9762:79,57,0
OP597571.1      528     .       A       C       35.38   PASS    F       GT:GQ:DP:AD:AF:PL       1:35:44:0,41:0.9318:80,63,0
OP597571.1      540     .       G       C       26.34   PASS    P       GT:GQ:DP:AD:AF:PL       1:26:45:0,44:0.9778:80,54,0
OP597571.1      741     .       A       .       23.27   RefCall P       GT:GQ:DP:AD:AF:PL       0:23:49:30:0.6122:990
OP597571.1      747     .       A       G       24.68   PASS    F       GT:GQ:DP:AD:AF:PL       1:24:47:4,40:0.8511:74,37,0
OP597571.1      888     .       T       C       24.92   PASS    P       GT:GQ:DP:AD:AF:PL       1:24:58:1,56:0.9655:80,42,0
OP597571.1      1335    .       G       A       28.63   PASS    F       GT:GQ:DP:AD:AF:PL       1:28:43:0,40:0.9302:77,45,0
OP597571.1      1469    .       A       G       30.17   PASS    F       GT:GQ:DP:AD:AF:PL       1:30:52:2,49:0.9423:80,48,0
OP597571.1      1566    .       G       A       32.47   PASS    F       GT:GQ:DP:AD:AF:PL       1:32:54:2,52:0.9630:80,53,0
OP597571.1      1677    .       T       C       31.74   PASS    F       GT:GQ:DP:AD:AF:PL       1:31:57:4,49:0.8596:80,52,0
OP597571.1      1806    .       G       A       27.10   PASS    P       GT:GQ:DP:AD:AF:PL       1:27:58:0,54:0.9310:80,47,0
OP597571.1      2051    .       T       C       26.04   PASS    P       GT:GQ:DP:AD:AF:PL       1:26:168:1,156:0.9286:80,41,0
OP597571.1      2265    .       G       .       .       .       .       GT      ./.

full_alignment.vcf.gz:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  SAMPLE
OP597571.1      185     .       G       A       9.65    PASS    F       GT:GQ:DP:AD:AF:PL       1/1:9:143:2,107:0.7483:18,13,0
OP597571.1      510     .       C       T       33.42   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:33:42:0,41:0.9762:79,57,0
OP597571.1      528     .       A       C       35.38   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:35:44:0,41:0.9318:80,63,0
OP597571.1      747     .       A       G       24.68   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:24:47:4,40:0.8511:74,37,0
OP597571.1      1335    .       G       A       28.63   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:28:43:0,40:0.9302:77,45,0
OP597571.1      1469    .       A       G       30.17   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:30:52:2,49:0.9423:80,48,0
OP597571.1      1566    .       G       A       32.47   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:32:54:2,52:0.9630:80,53,0
OP597571.1      1677    .       T       C       31.74   PASS    F       GT:GQ:DP:AD:AF:PL       1/1:31:57:4,49:0.8596:80,52,0

pileup.vcf.gz:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  SAMPLE
OP597571.1      185     .       G       A       19.96   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:19:143:2,107:0.7483:60,28,0
OP597571.1      252     .       C       A       25.12   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:25:90:1,84:0.9333:80,58,0
OP597571.1      510     .       C       T       21.82   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:21:42:0,41:0.9762:79,31,0
OP597571.1      528     .       A       C       24.57   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:24:44:0,41:0.9318:80,43,0
OP597571.1      540     .       G       C       26.34   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:26:45:0,44:0.9778:80,54,0
OP597571.1      741     .       A       .       23.27   RefCall P       GT:GQ:DP:AD:AF:PL       0/0:23:49:30:0.6122:990
OP597571.1      747     .       A       G       20.39   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:20:47:4,40:0.8511:80,28,0
OP597571.1      888     .       T       C       24.92   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:24:58:1,56:0.9655:80,42,0
OP597571.1      1335    .       G       A       22.79   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:22:43:0,40:0.9302:80,32,0
OP597571.1      1469    .       A       G       24.83   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:24:52:2,49:0.9423:80,37,0
OP597571.1      1566    .       G       A       22.71   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:22:54:2,52:0.9630:80,32,0
OP597571.1      1677    .       T       C       24.33   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:24:57:4,49:0.8596:80,38,0
OP597571.1      1806    .       G       A       27.10   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:27:58:0,54:0.9310:80,47,0
OP597571.1      2051    .       T       C       26.04   PASS    P       GT:GQ:DP:AD:AF:PL       1/1:26:168:1,156:0.9286:80,41,0

[aquaskyline]

Clair3 was seeing no read at 2265. Clair3 filters the alignments with the following four flags:
read unmapped (0x4)
mate unmapped (0x8)
not primary alignment (0x100)
supplementary alignment (0x800)
Using samtools, the alignments can be filtered with samtools view -F 2316, and then piped into bcftools mpileup. Please test if 2265 can still be called using mpileup after applying the filters.

[peterk87]

I filtered the alignments using samtools view -F 2316, however, the Bcftools results are mostly unchanged with only the depths and observed allele counts coming in a little lower.

Filtered BAM bcftools call results:

OP597571.1      2265    .       G       A       228.399 .       DP=759;VDB=0;SGB=-0.693147;RPBZ=-0.492301;MQBZ=1.61067;MQSBZ=3.01461;BQBZ=6.9304;SCBZ=0.520211;MQ0F=0;AC=1;AN=1;DP4=22,0,684,45;MQ=54   GT:PL   1:255,0

Unfiltered BAM bcftools call results:

OP597571.1      2265    .       G       A       228.4   .       DP=802;VDB=0;SGB=-0.693147;RPBZ=-0.477099;MQBZ=2.06864;MQSBZ=3.44984;BQBZ=7.12028;SCBZ=0.475257;MQ0F=0;AC=1;AN=1;DP4=23,0,710,60;MQ=54  GT:PL   1:255,0

Is there anything else that might be going on?

[aquaskyline]

That's challenging. To make things easier, would you mind sending us a minibam with reads covering 2265.

[peterk87]

Sure thing! Please find attached the samtools view filtered BAM:

filt.bam.gz

Let me know if you need anything else!

[aquaskyline]

We found that 2265 is the last 16th base in your reference. Clair3 doesn't call variants in the first 16bp and last 16bp of a sequence because of 1) algorithmic limit, and 2) usually degenerated coverage and alignment performance in the head and tail of a sequence that makes variant calling unreliable. A solution to your case is to add say 10 'N' to the tail of your reference genome so 2265 gets out of the tail 16bp limit.

[peterk87]

Thanks. This is very good to know.

For Influenza virus sequencing, there's a set of primers targeting the conserved 5' and 3' ends of each of the 8 genome segment generating amplicons that result in good sequencing coverage even at the ends of each segment.

[aquaskyline]

Understood. Leaving this issue open and will come back later with a better solution.

[lagphase]

Clair3 was seeing no read at 2265. Clair3 filters the alignments with the following four flags: read unmapped (0x4) mate unmapped (0x8) not primary alignment (0x100) supplementary alignment (0x800) Using samtools, the alignments can be filtered with samtools view -F 2316, and then piped into bcftools mpileup. Please test if 2265 can still be called using mpileup after applying the filters.

Hello,

What is Clair3 filter criteria for supplementary alignments? I see mismatches in IGV that were not called as SNP by Clair3. I clicked on the reads and almost most of them are supplementary alignments.

[zhengzhenxian]

@lagphase By default, Clair3 would discard all supplementary alignments. So you might disable the supplementary alignments in IGV to see if the variant is evident.

[aquaskyline]

New code was added to Clair3-RNA to take care of the head/tail 16bp of each sequence, and is now being tested. The same function solving this issue will be introduced to Clair3 in the next release.