Feature for allele search doesn't intersect JGene error when running findAlleles

[michael-ford]

Checklist before submitting the issue:

• The issue is strongly related to the MiXCR software
• The issue can be reproduced with the most recent version of MiXCR
• There is no answer to the question in the official documentation and there is no duplicate issue in the bug tracker
• Inspection of raw alignments with exportAlignmentsPretty shows that data has the expected architecture, and sample preparation artefacts are not the reason of the problem (if this is the matter of the issue)

Expected Result

Have run mixcr analyze invivoscribe-human-dna-ighv-leader-lymphotrack, now trying to run findAlleles to call alleles / novel variants. Getting a Feature for allele search doesn't intersect JGene error?

Confirmed the following:

1. clns file has clones by running mixcr exportAirr - all clones seem to have V, D and J calls. Example:

clone_id        sequence_id     sequence        rev_comp        productive      v_call  d_call  j_call  c_call  sequence_alignment      germline_alignment      complete_vdj    junction        junction_aa     np1     np2     cdr1    cdr1_aa cdr2    cdr2_aa cdr3    cdr3_aa fwr1    fwr1_aa fwr2    fwr2_aa fwr3    fwr3_aa fwr4    fwr4_aa v_score v_cigar d_score d_cigar   j_score j_cigar c_score c_cigar junction_length np1_length      np2_length      v_germline_start        v_sequence_start        v_germline_end  v_sequence_end  d_germline_start        d_sequence_start        d_germline_end  d_sequence_end  j_germline_start        j_sequence_start        j_germline_end  j_sequence_end  c_germline_start        c_sequence_start  c_germline_end  c_sequence_end  v_alignment_start       v_alignment_end d_alignment_start       d_alignment_end j_alignment_start       j_alignment_end c_alignment_start       c_alignment_end duplicate_count
0       clone.0 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACATGCACTGTCTGTGGTGACTCCATCAGTAGTTACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTCTTACACTGGGAGCACCACCTACAACCCCTCCCTCAAGAGTCGAGTCTCCATATCAATAGACACGTCCAAGAACCAGTTCTCCCTGAACCTGAGGTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAGAGGTGTATGTTAATGACTACTACTACGGTATGGACGTCTGG   F       TIGHV4-59*00      IGHD2-8*00      IGHJ6*00                CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACATGCACTGTCTGTGGTGACTCCATCAGTAGTTACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTCTTACACTGGGAGCACCACCTACAACCCCTCCCTCAAGAGTCGAGTCTCCATATCAATAGACACGTCCAAGAACCAGTTCTCCCTGAACCTGAGGTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAGAGGTGTATGTTAATGACTACTACTACGGTATGGACGTCTGG     CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAGGTGTATGCTANNNACTACTACTACGGTATGGACGTCTGG     F       TGTGCGAGAGAGGTGTATGTTAATGACTACTACTACGGTATGGACGTCTGG     CAREVYVNDYYYGMDVW               ATG     GGTGACTCCATCAGTAGTTACTAC        GDSISSYY        ATCTCTTACACTGGGAGCACC   ISYTGST GCGAGAGAGGTGTATGTTAATGACTACTACTACGGTATGGACGTC   AREVYVNDYYYGMDV CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACATGCACTGTCTGT     QVQLQESGPGLVKPSETLSLTCTVCTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATAT      WNWIRQPPGKGLEWIGY       ACCTACAACCCCTCCCTCAAGAGTCGAGTCTCCATATCAATAGACACGTCCAAGAACCAGTTCTCCCTGAACCTGAGGTCTGTGACCGCTGCGGACACGGCCGTGTATTAT TYNPSLKSRVSISIDTSKNQFSLNLRSVTAADTAVYY                   2553.0  159N62=1X10=1X5=1X23=1X42=1X7=1X5=1X11=1X28=1X8=1X31=1X5=1X32=1X11=     41.0    293S47N8=1X2=   260.0   307S26N26=                        51      0       3       160     1       452     293     48      294     58      304     27      308     52      333                                     1       293     294     304     308     333                     37340

2. exportAlignmentsPretty shows reads covering L1 through FR4:

>>> Read ids: 0


               ><L1
                <5'UTR                                     L1>
                 M  K  H  L  W  F  F  L  L  L  V  A  A  P  R  w
    Quality    22622267777777777777777777777776777777777777777777777777777767777777777777777777
    Target0  0 TATGAAGCACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGTGAGTATCTCAGGGATCCAGACATGGGGATAT 79  Score
IGHV4-59*00 20  atgaaAcaTctgtggttcttccttctcctggtggcagctcccagatgtgagtatctcagggatccagacatggggatat 98  3856

                                                                <L2     L2><FR1
                                                               w  V  L  S  Q  V  Q  L  Q  E  S
    Quality    77777777777777777777777777777777777777777777777777777777776777777777777777777777
    Target0 80 GGGAGGTGCCTCTGATCCCAGGGCTCACTGTGGGTCTCTCTGTTCACAGGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTC 159  Score
IGHV4-59*00 99 gggaggtgcctctgatcccagggctcactgtgggtctctctgttcacaggggtcctgtcccaggtgcagctgcaggagtc 178  3856

                                                                   FR1><CDR1              CDR1><
                 G  P  G  L  V  K  P  S  E  T  L  S  L  T  C  T  V  S  G  D  S  I  S  R  Y  N  W
    Quality     77777777777777777777777776567767777777775777674777777777737677777577777567767777
    Target0 160 GGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACATGCACTGTCTCTGGTGACTCCATCAGTAGGTACAACT 239  Score
IGHV4-59*00 179 gggcccaggactggtgaagccttcggagaccctgtccctcacCtgcactgtctctggtgGctccatcagtagTtacTact 258  3856

                                                              FR2><CDR2           CDR2><FR3
                  R  W  I  R  Q  P  P  G  K  G  L  E  W  I  G  Y  I  S  Y  T  G  S  T  T  Y  N
    Quality     77777655353577677777776735765555366737766576675767777766767756545675511764567657
    Target0 240 GGAGGTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTCTTACACTGGGAGCACCACCTACAAC 319  Score
IGHV4-59*00 259 ggagCtggatccggcagcccccagggaagggactggagtggattggGtatatctAttacaGtgggagcaccaActacaac 338  3856


                P  S  L  K  S  R  V  S  I  S  I  D  T  S  K  N  Q  F  S  L  N  L  R  S  V  T  A
    Quality     76167467776777766676222777677757777777767677777777776646777777776777777677777777
    Target0 320 CCCTCCCTCAAGAGTCGAGTCTCCATATCAATAGACACGTCCAAGAACCAGTTCTCCCTGAACCTGAGGTCTGTGACCGC 399  Score
IGHV4-59*00 339 ccctccctcaagagtcgagtcAccatatcaGtagacacgtccaagaaccagttctccctgaaGctgagCtctgtgaccgc 418  3856

                                  FR3><CDR3    V><D       D>  <J                    CDR3><FR4
                 A  D  T  A  V  Y  Y  C  A  R  E  V  Y  V  N  Y  Y  Y  Y  G  M  D  V  W   G  Q
    Quality     47777677777777777777775777777777777777777777777777777777777777777777777777777777
    Target0 400 TGCGGACACGGCCGTGTATTATTGTGCGAGAGAGGTGTATGTTAATTACTACTACTACGGTATGGACGTCTGGGGCCAAG 479  Score
IGHV4-59*00 419 tgcggacacggccgtgtattaCtgtgcgagaga                                                451  3856
 IGHD2-8*00  47                                  ggtgtatgCta                                     57   41
   IGHJ6*00  25                                               tactactactacggtatggacgtctggggccaag 58   580

                                 FR4>
             G  T  T  V  T  V  S  S _
 Quality     777777777777777777777766646
 Target0 480 GGACCACGGTCACCGTCTCCTCAGGTA 506  Score
IGHJ6*00  59 ggaccacggtcaccgtctcctcag    82   580

Actual Result

$ mixcr findAlleles --output-template {file_name}.allelic.clns --export-alleles-mutations allele_stats.tsv --verbose  02-RE13-BLD-Leader_S2_L001.clns
Available CPU: 16, used CPU: unspecified. Available memory: 40960 Mb, used memory: 12000 Mb
Feature for search alleles: {FR1Begin:CDR3End}
Name of the original library: repseqio.v4.0
Step 1 of 7: count diversity for dataset: 0%
App version: 4.6.0; built=Sat Dec 09 14:48:42 EST 2023; rev=c9fafa41fe; lib=repseqio.v4.0
com.milaboratory.app.ValidationException: Feature for allele search doesn't intersect JGene
        at com.milaboratory.o.gP.<init>(SourceFile:122)
        at com.milaboratory.o.gP$b.a(SourceFile:1176)
        at com.milaboratory.mixcr.cli.CommandFindAlleles.run1(SourceFile:353)
        at com.milaboratory.mixcr.cli.MiXCRCommandWithOutputs.run0(SourceFile:69)
        at com.milaboratory.mixcr.cli.MiXCRCommand.run(SourceFile:37)
        at picocli.CommandLine.executeUserObject(CommandLine.java:1939)
        at picocli.CommandLine.access$1300(CommandLine.java:145)
        at picocli.CommandLine$RunLast.executeUserObjectOfLastSubcommandWithSameParent(CommandLine.java:2358)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2352)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2314)
        at picocli.CommandLine$AbstractParseResultHandler.execute(CommandLine.java:2179)
        at picocli.CommandLine$RunLast.execute(CommandLine.java:2316)
        at com.milaboratory.mixcr.cli.Main.registerLogger$lambda-27(SourceFile:514)
        at picocli.CommandLine.execute(CommandLine.java:2078)
        at com.milaboratory.mixcr.cli.Main.main(SourceFile:101)
Feature for allele search doesn't intersect JGene
com.milaboratory.app.ValidationException: Feature for allele search doesn't intersect JGene
        at com.milaboratory.o.gP.<init>(SourceFile:122)
        at com.milaboratory.o.gP$b.a(SourceFile:1176)
        at com.milaboratory.mixcr.cli.CommandFindAlleles.run1(SourceFile:353)
        at com.milaboratory.mixcr.cli.MiXCRCommandWithOutputs.run0(SourceFile:69)
        at com.milaboratory.mixcr.cli.MiXCRCommand.run(SourceFile:37)
        at picocli.CommandLine.executeUserObject(CommandLine.java:1939)
        at picocli.CommandLine.access$1300(CommandLine.java:145)
        at picocli.CommandLine$RunLast.executeUserObjectOfLastSubcommandWithSameParent(CommandLine.java:2358)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2352)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2314)
        at picocli.CommandLine$AbstractParseResultHandler.execute(CommandLine.java:2179)
        at picocli.CommandLine$RunLast.execute(CommandLine.java:2316)
        at com.milaboratory.mixcr.cli.Main.registerLogger$lambda-27(SourceFile:514)
        at picocli.CommandLine.execute(CommandLine.java:2078)
        at com.milaboratory.mixcr.cli.Main.main(SourceFile:101)

Exact MiXCR commands

mixcr findAlleles --output-template {file_name}.allelic.clns --export-alleles-mutations allele_stats.tsv --verbose  02-RE13-BLD-Leader_S2_L001.clns

[mizraelson]

Hi, because for this kit the reverse primer is located in FR4 region, the clones are assembled by {FR1Begin:CDR3End} feature to exclude the primer sequence. If needed you can manually add the following parameter to mixcr analyze command and then findAlleles would work:

mixcr analyze invivoscribe-human-dna-ighv-leader-lymphotrack \
--assemble-clonotypes-by VDJRegion \
input_R1.fastq.gz \
input_R2.fastq.gz \
output