nMutationRate in Somatic Hyper Mutation tree

[singhd6]

Hi MIXCR Team,
I have a question regarding nMutation rate in somatic hypermutation tree in mixcr. I want to understand the following phenomenon. Could anyone please help me with my question.
Thank you so much in advance for your time and effort.
I generated the SHMT trees with following commands for mouse BCR sequencing:
#Find alleles#
mixcr findAlleles
--report "/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2.findAlleles.report.txt"
--export-alleles-mutations "/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2/PANPAN2_Alleles.tsv"
--export-library "/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2/PANPAN2_Alleles.json"
--output-template "/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/result2/Pan-pan2.reassigned.clns"
"/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/result2/Pan-pan2S12.clns"

#Export the reassigned .clns in .shmt format#
mixcr findShmTrees
--report "/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/PAN-PAN2/PANPAN2_trees.log"
"/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/result2/Pan-pan2.reassigned.clns"
"/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2.shmt"
#export SHM trees with nodes#
mixcr exportShmTreesWithNodes
"/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2.shmt"
"/mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/Pan-pan2S12/SHMT_PAN-PAN2/PANPAN2_Trees.tsv"

I am trying to understand the somatic hypermutation trees:
treeId nodeId isObserved chains parentId DistanceFromGermline nMutationsRate cloneId fileName readCount readFraction targetSequences targetQualities bestVHit bestDHit bestJHit bestCHit allVAlignments allDAlignments allJAlignments allCAlignments nSeqFR1 nSeqCDR1 nSeqFR2 nSeqCDR2 nSeqFR3 nSeqCDR3 nSeqFR4 aaSeqFR1 aaSeqCDR1 aaSeqFR2 aaSeqCDR2 aaSeqFR3 aaSeqCDR3 aaSeqFR4 isotype refPoints
74 14 TRUE IGH 4 132 0.390532544 3233 /mnt/beegfs/singhd6/Divya_data/vhseq/BCR_Seq_Mouse_GZ/JA10S1/JA10SHMT/JA10.reassigned.clns 124 2.88E-05 CTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGTATCTATGATGGTTACTTCCACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-100-M8-5OIS4G6YYQRS2JEG37HKSY5G IGHD2-300 IGHJ200 IGHM00 74|367|388|0|161|DG74DA75DG76DG77DT78DG79DA80DA81DG82DC83DT84DT85DC86DT87DC88DG89DA90DG91DT92DC93DT94DG95DG96DA97DG98DG99DT100DG101DG102DC103DC104DT105DG106DG107DT108DG109DC110DA111DG112DC113DC114DT115DG116DG117DA118DG119DG120DA121DT122DC123DC124DC125DT126DG127DA128DA129DA130DC131DT132DC133DT134DC135DC136DT137DG138DT139DG140DC141DA142DG143DC144DC145DT146DC147DA148DG149DG150DA151DT152DT153DC154DG155DA156DT157DT158DT159DT160DA161DG162DT163DA164DG165DA166DT167DA168DC169DT170DG171DG172DA173DT174DG175DA176DG177DT178DT179DG180DG181DG182DT183DC184DC185DG186DG187DC188DA189DG190DG191DC192DT193DC194DC195DA196DG197DG198DG199DA200DA201DA202DG203DG204DG205|107.0 16|32|51|162|178||80.0 23|68|68|181|226||450.0 CTAGAATGGATTGGAGAA ATTAATCCAGATAGCAGTACGATA AACTATACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTAC TGTGCAAGTATCTATGATGGTTACTTCCACTTTGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCAG LEWIGE INPDSSTI NYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYY CASIYDGYFHFDYW GQGTTLTVSS IgM ::::0:0:0:18:42:153:-1:161:162:1:-2:178:181:-3:195:226::
"In the above tree the nMutation rate for treeID74 is 0.390532544 and distance from germline is 132, according to MIXCR documents: n mutation rate is "Number of nucleotide mutations from germline divided by target sequence size" now if i see the target sequence size of the treeID74 is 226 and i divide 132/226 = 0.584070796460177, then why the nMuatation rate is 0.390532544. Unless target sequence size is something else and how mixcr is calculating the mutation rate i want to understand. I calculated the total number of nucleotide from the column target sequence which is 226. Can you please explain how do you calculate the nMutation rate for somatic hypermutation tree."
Thank you
Divya

[mizraelson]

The thing is, mutations are also calculated for the CDR3 region. Additionally, wildcard resolutions in the VJJunction are excluded from this calculation. However, in the distance calculation, wildcards are taken into account — which is why the value can appear larger.

Could you try exporting the following column as well and see if the value is closer?

-nFeature VDJRegion mrca

[singhd6]

Hi Mark,
Thank you so much for your help. Yes when I exported the target sequences with VDJregion mrca now the mutation rate is correct and matches as in the shm trees. I really appreciate your timely response.
Thank you again.
Divya