mizraelson
commented
2 months ago Yes, the BCR gene segments often have very similar sequences. If your reads do not cover the entire gene sequence, there might be several candidates. In the brackets following the gene names, you see the alignment scores. The first gene in the list is the most probable one with the highest score.
i run the code just as following: mixcr align -Xmx15g -p rna-seq -s hsa -OallowPartialAlignments=true ./f1.rmrrna.fastp.fastq.1.gz ./f1.rmrrna.fastp.fastq.2.gz ./f1.vdjca; mixcr assemblePartial -Xmx15g ./f1.vdjca ./f1.alignments_rescued_1.vdjca mixcr assemblePartial -Xmx15g ./f1.alignments_rescued_1.vdjca ./f1.alignments_rescued_2.vdjca mixcr extend -Xmx15g ./f1.alignments_rescued_2.vdjca ./f1.alignments_rescued_2_extended.vdjca mixcr assemble -Xmx15g ./f1.alignments_rescued_2_extended.vdjca ./f1.clones.clns mixcr exportClones -Xmx15g ./f1.clones.clns ./f1.clones.txt; done mixcr exportClones -Xmx15g -c IGH ./f1.clones.clns ./f1.IGH.clones.txt
After that, f1.IGH.clones.txt results shown that some reads could mapping to multi-VDJ. is this is normal？
cloneId | cloneCount | cloneFraction | targetSequences | targetQualities | allVHitsWithScore | allDHitsWithScore | allJHitsWithScore | allCHitsWithScore | allVAlignments | allDAlignments | allJAlignments | allCAlignments | nSeqFR1 | minQualFR1 | nSeqCDR1 | minQualCDR1 | nSeqFR2 | minQualFR2 | nSeqCDR2 | minQualCDR2 | nSeqFR3 | minQualFR3 | nSeqCDR3 | minQualCDR3 | nSeqFR4 | minQualFR4 | aaSeqFR1 | aaSeqCDR1 | aaSeqFR2 | aaSeqCDR2 | aaSeqFR3 | aaSeqCDR3 | aaSeqFR4 | refPoints -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- 3 | 28 | 0.019270475 | TGTGGATCAGATGCCAGAGCACCAGACGGCAATGGTTATCACTATGCTTTTGCTATGTGG | NNNNNNNNNNNNNNNNNNNN???????????????????????????????????????? | IGHV1-69-2*00(249.2) | IGHD3-22*00(39),IGHD3-3*00(35),IGHD5-18*00(35) | IGHJ3*00(156.5) | IGHG1*00(459),IGHG3*00(402.8) | 359\|371\|390\|0\|12\|SC363GSA365T\|28.0 | 49\|60\|93\|32\|43\|ST56C\|39.0;49\|56\|93\|32\|39\|\|35.0;32\|39\|60\|31\|38\|\|35.0 | 22\|39\|70\|43\|60\|SA31CSC35G\|53.0 | ; | | | | | | | | | | | TGTGGATCAGATGCCAGAGCACCAGACGGCAATGGTTATCACTATGCTTTTGCTATGTGG | 30 | | | | | | | | CGSDARAPDGNGYHYAFAMW | | :::::::::0:1:12:32:-18:-2:43:43:-2:60::: 14 | 12 | 0.008258775 | TGTGCGAGAGTCGGGTACCGTGGTGGTGCCACACCTTTTGACTACTGG | ???????????????????????????????????????????????? | IGHV4-39*00(337.6) | IGHD2-21*00(45),IGHD2-15*00(40) | IGHJ4*00(200) | IGHM*00(372.3) | 445\|454\|476\|0\|9\|\|45.0 | 36\|45\|84\|19\|28\|\|45.0;42\|50\|93\|19\|27\|\|40.0 | 25\|37\|68\|36\|48\|\|60.0 | | | | | | | | | | | | TGTGCGAGAGTCGGGTACCGTGGTGGTGCCACACCTTTTGACTACTGG | 30 | | | | | | | | CARVGYRGGATPFDYW | | :::::::::0:-2:9:19:-8:-11:28:36:-5:48::: 33 | 8 | 0.00550585 | TGTGCGCGACATCGGGCAGTCCCGGAGTTCGGGGAGGGACTTGACTATTGG | ??????????????????????????????????????????????????? | IGHV1-46*00(73.5),IGHV3-53*00(72.3) | IGHD3-10*00(50) | IGHJ4*00(174) | IGHG2*00(832.8) | 442\|455\|473\|0\|13\|SA448CSG451C\|33.0;439\|452\|470\|0\|13\|SA445CSG448C\|33.0 | 42\|52\|93\|26\|36\|\|50.0 | 26\|37\|68\|40\|51\|SC33T\|39.0 | | | | | | | | | | | | TGTGCGCGACATCGGGCAGTCCCGGAGTTCGGGGAGGGACTTGACTATTGG | 30 | | | | | | | | CARHRAVPEFGEGLDYW | | :::::::::0:2:13:26:-11:-10:36:40:-6:51::: 34 | 8 | 0.00550585 | TGTGCAAGAGATTCCGGAAACATTTATGGCGCGTACTACTTTGATTCCTGG | ??????????????????????????????????????????????????? | IGHV6-1*00(340.4) | IGHD2-21*00(35),IGHD2-15*00(30),IGHD4-23*00(30) | IGHJ4*00(200) | IGHG2*00(369.4) | 454\|466\|485\|0\|12\|\|60.0 | 53\|60\|84\|12\|19\|\|35.0;59\|65\|93\|12\|18\|\|30.0;35\|41\|57\|12\|18\|\|30.0 | 18\|37\|68\|32\|51\|SC30TSA32C\|63.0 | | | | | | | | | | | | TGTGCAAGAGATTCCGGAAACATTTATGGCGCGTACTACTTTGATTCCTGG | 30 | | | | | | | | CARDSGNIYGAYYFDSW | | :::::::::0:1:12:12:-25:4:19:32:2:51::: 38 | 7 | 0.004817619 | TGTGCGAGAGAATCGACTGAGACATGGCTACAAGTGTCCAACTATTTTGAACACTGG | ????????????????????????????????????????????????????????? | IGHV1-2*00(287.3) | IGHD5-24*00(54) | IGHJ4*00(162.8),IGHJ5*00(145.8) | IGHA1*00(527.5) | 442\|453\|473\|0\|11\|\|55.0 | 22\|36\|60\|19\|33\|SG25C\|54.0 | 20\|37\|68\|40\|57\|SC24TSC30AST31C\|37.0;36\|40\|71\|53\|57\|\|20.0 | | | | | | | | | | | | TGTGCGAGAGAATCGACTGAGACATGGCTACAAGTGTCCAACTATTTTGAACACTGG | 30 | | | | | | | | CARESTETWLQVSNYFEHW | | :::::::::0:0:11:19:-2:-4:33:40:0:57:::