Error during creating the SHMtree plot

[singhd6]

Hi I am running the follwoing command and I receive the error of "Unmatched arguments from index 12":
Script I ran:
mixcr exportPlots shmTrees
-m "/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/metadata.tsv"
--node-color Timepoint
--line-color Subset
--ids 29,49,79
"/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/E2-SUBJ059-IgM_S13_L001.shmt"
"/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/SUBJ001_IgG_A_S3.shmt"
"/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/B1-SUBJ027-IgG-A_S2.shmt"
"/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/C2-SUBJ053-IgM_S11_L001.shmt"
"/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/figs1/IM_shm_trees.pdf"

Error:
Unmatched arguments from index 12: '/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/B CR_SEQUENCING_DS_REANALYSIS/lineage_tree/B1-SUBJ027-IgG-A_S2.shmt', '/mnt/beegfs/singhd6/ Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_REANALYSIS/lineage_tree/C2-SUBJ053-IgM_ S11_L001.shmt', '/mnt/beegfs/singhd6/Divya_data/vhseq/mixcr_analysis/BCR_SEQUENCING_DS_RE ANALYSIS/lineage_tree/figs1/IM_shm_trees.pdf'

Could you please help me with the issue ?

Thank you so much in advance
Divya

[mizraelson]

Hi, mixcr exportPlots shmTrees can only accept a single .shmt file as input. Why are you trying to add multiple files? Also, I highly recommend trying our new software, Paltforma—it offers significantly enhanced functionality for SHM tree visualization.

[singhd6]

Hi Mark, Thank you for replying to my issue. I have a question: what type of built-in gene libraries are used in VDJ annotation in Mixcr, I downloaded the built in gene libraries for VDJ annoatation, but the structure of the fasta file is different from the IMGT ? Thank you Divya

…

________________________________ From: mizraelson ***@***.***> Sent: Tuesday, March 4, 2025 12:04 AM To: milaboratory/mixcr ***@***.***> Cc: Singh, Divya Jyoti ***@***.***>; Author ***@***.***> Subject: [EXT] Re: [milaboratory/mixcr] Error during creating the SHMtree plot (Discussion #1910) PROCEED WITH CAUTION: Slow down and pay close attention to emails sent from outside the organization. If you receive an unsolicited email from an unknown sender or are suspicious of the tone, style, vocabulary or urgency of the email message, never click links or open attachments within it. When in doubt, you should either delete the email, verify its authenticity by contacting the sender using an alternative method not listed in the email, or submit it via the BlueFish button in Outlook for investigation. If you don't have the BlueFish button or are using a mobile device, forward the email as an attachment to ***@***.******@***.***?subject=Report%20a%20Suspicious%20Email>

________________________________ Hi, mixcr exportPlots shmTrees can only accept a single .shmt file as input. Why are you trying to add multiple files? Also, I highly recommend trying our new software, Paltforma<https://platforma.bio/>—it offers significantly enhanced functionality for SHM tree visualization. — Reply to this email directly, view it on GitHub<#1910 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BH7E6ZJ4NVYDQWBODTYROIL2SUX7HAVCNFSM6AAAAABYEPLTX6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTEMZYGM3TKMQ>. You are receiving this because you authored the thread.Message ID: ***@***.***> Please consider the environment before printing this e-mail Cleveland Clinic is a nonprofit, multispecialty academic medical center that's recognized in the U.S. and throughout the world for its expertise and care. Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you.

[mizraelson]

MiXCR uses its own library format. Are you trying to create a custom reference or use the IMGT one?

[singhd6]

Dear Mark, Good morning, I am trying to format the clones with create Germlines function of R-Immcantation Shazam and Dowser tools. And for the said purpose, I want to run the following command with AIRR format clone tables. But since the reference library is different than the IMGT while creating the clonotype in MIXCR all the alleles are filtered after creating the germline in dowser. I want to analyze the detailed mutational load during the clonal evolution whether it is replacement mutation or silent mutation what the frequencies of mutation among different subjects, mutation due to antigen selection pressure. And all these detailed mutations analysis could be done in R. I downloaded the VDJ gene libraries from https://vdj.online/library Is this the correct gene libraries from where MIXCR annotate clones? VDJ.online | Align with MiXCR<https://vdj.online/library> Map raw sequencing reads to the verified reference database of TCR/IG gene segments to get exhaustive information on VDJ markup, mutations and antibody isotype. vdj.online library(dowser) library(dplyr) library(alakazam) library(dplyr) library(ggplot2) library(scales) library(readr) library(pheatmap) library(airr) library(devtools) ***@***.***") library(enchantr) library(stringr) library(shazam) loaded_data <- readInput("C:/Users/singhd6/Documents/Shazam_Test_Data", pattern="AIRR.tsv") references = readIMGT(dir = file.path("C:/Users/singhd6/Documents/germlines/human/vdj")) loaded_data$sequence_id <- paste0(loaded_data$sequence_id, "_", loaded_data$sample_id) createGermlines( loaded_data, references, locus = "locus", trim_lengths = FALSE, force_trim = FALSE, nproc = 1, seq = "sequence_id", v_call = "v_call", d_call = "d_call", j_call = "j_call", amino_acid = FALSE, id = "sequence_id", clone = "clone_id", v_germ_start = "v_germline_start", v_germ_end = "v_germline_end", v_germ_length = "v_germline_length", d_germ_start = "d_germline_start", d_germ_end = "d_germline_end", d_germ_length = "d_germline_length", j_germ_start = "j_germline_start", j_germ_end = "j_germline_end", j_germ_length = "j_germline_length", np1_length = "np1_length", np2_length = "np2_length", na.rm = TRUE, fields = NULL, verbose = 0) Error: # A tibble: 0 × 73 # ℹ 73 variables: clone_id <chr>, sequence_id <chr>, sequence <chr>, rev_comp <lgl>, productive <lgl>, v_call <chr>, d_call <chr>, # j_call <chr>, c_call <chr>, sequence_alignment <chr>, germline_alignment <lgl>, complete_vdj <lgl>, junction <chr>, # junction_aa <chr>, np1 <chr>, np2 <chr>, cdr1 <chr>, cdr1_aa <chr>, cdr2 <chr>, cdr2_aa <chr>, cdr3 <chr>, cdr3_aa <chr>, # fwr1 <chr>, fwr1_aa <chr>, fwr2 <chr>, fwr2_aa <chr>, fwr3 <chr>, fwr3_aa <chr>, fwr4 <chr>, fwr4_aa <chr>, v_score <dbl>, # v_cigar <chr>, d_score <dbl>, d_cigar <chr>, j_score <dbl>, j_cigar <chr>, c_score <dbl>, c_cigar <chr>, junction_length <int>, # np1_length <int>, np2_length <int>, v_germline_start <int>, v_sequence_start <int>, v_germline_end <int>, v_sequence_end <int>, # d_germline_start <int>, d_sequence_start <int>, d_germline_end <int>, d_sequence_end <int>, j_germline_start <int>, … There were 50 or more warnings (use warnings() to see the first 50)

warnings()

Warning messages: 1: In createGermlines(loaded_data, references, locus = "locus", ... : locus column not found, attempting to extract locus from V call 2: In createGermlines(loaded_data, references, locus = "locus", ... : Loci found: IGH 3: In getGermline(receptor, references$V, segment = "V", ... : Allele IGHV4-3400 is not in the provided germline database. 4: In getGermline(receptor, references$D, segment = "D", ... : Allele IGHD3-300 is not in the provided germline database. 5: In getGermline(receptor, references$J, segment = "J", ... : Allele IGHJ400 is not in the provided germline database. 6: In getGermline(receptor, references$V, segment = "V", ... : Allele IGHV4-3400 is not in the provided germline database. 7: In getGermline(receptor, references$D, segment = "D", ... : Allele IGHD2-800 is not in the provided germline database. Thank you Divya

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________________________________ From: mizraelson ***@***.***> Sent: Wednesday, March 5, 2025 4:19 PM To: milaboratory/mixcr ***@***.***> Cc: Singh, Divya Jyoti ***@***.***>; Author ***@***.***> Subject: [EXT] Re: [milaboratory/mixcr] Error during creating the SHMtree plot (Discussion #1910) PROCEED WITH CAUTION: Slow down and pay close attention to emails sent from outside the organization. If you receive an unsolicited email from an unknown sender or are suspicious of the tone, style, vocabulary or urgency of the email message, never click links or open attachments within it. When in doubt, you should either delete the email, verify its authenticity by contacting the sender using an alternative method not listed in the email, or submit it via the BlueFish button in Outlook for investigation. If you don't have the BlueFish button or are using a mobile device, forward the email as an attachment to ***@***.******@***.***?subject=Report%20a%20Suspicious%20Email>

________________________________ MiXCR uses its own library format. Are you trying to create a custom reference or use the IMGT one? — Reply to this email directly, view it on GitHub<#1910 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BH7E6ZKPMTYP7XJM4UHAU2T2S5S6PAVCNFSM6AAAAABYEPLTX6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTENBQGY2TSNI>. You are receiving this because you authored the thread. Please consider the environment before printing this e-mail Cleveland Clinic is a nonprofit, multispecialty academic medical center that's recognized in the U.S. and throughout the world for its expertise and care. Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you.

[mizraelson]

MiXCR can also output clonotypes in AIRR format if that is what you are looking for.
By default MiXCR does not use the allele reference, because many of the reported IMGT alleles are misleading. Instead MiXCR has a find allele feature to infer the allelic variants from your data given you have a sufficient number of clones.

Also, you cna add a germline to the regular MiXCR export:

-nFeature <gene_feature> [germline]
Export nucleotide sequence of specified gene feature. If second option is germline than will be exported corresponded sequence of the top gene. It's recommended to run findAlleles before exporting -nFeature <gene_feature> germline because otherwise germline sequence will not incorporate allelic mutations. For clones from all chains.

[singhd6]

Hi Mark, Thanks for your response. I have the AIRR format clonotype table from mixcr and also, I have generated the SHM table too. However, purpose of my analysis is deeper visualization of the somatic hypermutation events, and for the said purpose I am utilizing immcantation tools in R, but immcantation does not read the AIRR format clonotype table and then format the clone to create the germline alignments with the allele because of different allelic structures as mentioned. However, I am trying to create the lineage tree image in platforma, does platforma accept the AIRR TSV files or SHM tree files rather than starting from scratch FASTQ. Because I have already Generated the clonotype AIRR TSV and SHM trees from FASTQ. And generating the clonotype table and SHM tree table again is time and resources intensive task. Thank you Divya

…

________________________________ From: mizraelson ***@***.***> Sent: Thursday, March 6, 2025 12:43 PM To: milaboratory/mixcr ***@***.***> Cc: Singh, Divya Jyoti ***@***.***>; Author ***@***.***> Subject: [EXT] Re: [milaboratory/mixcr] Error during creating the SHMtree plot (Discussion #1910) PROCEED WITH CAUTION: Slow down and pay close attention to emails sent from outside the organization. If you receive an unsolicited email from an unknown sender or are suspicious of the tone, style, vocabulary or urgency of the email message, never click links or open attachments within it. When in doubt, you should either delete the email, verify its authenticity by contacting the sender using an alternative method not listed in the email, or submit it via the BlueFish button in Outlook for investigation. If you don't have the BlueFish button or are using a mobile device, forward the email as an attachment to ***@***.******@***.***?subject=Report%20a%20Suspicious%20Email>

________________________________ MiXCR can also output clonotypes in AIRR format<https://mixcr.com/mixcr/reference/mixcr-exportAirr/> if that is what you are looking for. By default MiXCR does not use the allele reference, because many of the reported IMGT alleles are misleading. Instead MiXCR has a find allele feature to infer the allelic variants<https://mixcr.com/mixcr/reference/mixcr-findAlleles/> from your data given you have a sufficient number of clones. Also, you cna add a germline to the regular MiXCR export<https://mixcr.com/mixcr/reference/mixcr-export/#common-fields>:

-nFeature <gene_feature> [germline] Export nucleotide sequence of specified gene feature. If second option is germline than will be exported corresponded sequence of the top gene. It's recommended to run findAlleles before exporting -nFeature <gene_feature> germline because otherwise germline sequence will not incorporate allelic mutations. For clones from all chains. — Reply to this email directly, view it on GitHub<#1910 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BH7E6ZP5XDYHXLEUCSUZYZL2TCCNDAVCNFSM6AAAAABYEPLTX6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTENBRG4YTMMQ>. You are receiving this because you authored the thread. Please consider the environment before printing this e-mail Cleveland Clinic is a nonprofit, multispecialty academic medical center that's recognized in the U.S. and throughout the world for its expertise and care. Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you.

[mizraelson]

At the moment Platforma can only start from FASTQ files but in a few weeks we will add a feature to upload repertoires from tsv tables.

[singhd6]

Hi, I am working with platforma and loaded my rawdata fastq files, do you have documentation available for platforma? Thank you Divya

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________________________________ From: mizraelson ***@***.***> Sent: Thursday, March 6, 2025 4:47 PM To: milaboratory/mixcr ***@***.***> Cc: Singh, Divya Jyoti ***@***.***>; Author ***@***.***> Subject: [EXT] Re: [milaboratory/mixcr] Error during creating the SHMtree plot (Discussion #1910) PROCEED WITH CAUTION: Slow down and pay close attention to emails sent from outside the organization. If you receive an unsolicited email from an unknown sender or are suspicious of the tone, style, vocabulary or urgency of the email message, never click links or open attachments within it. When in doubt, you should either delete the email, verify its authenticity by contacting the sender using an alternative method not listed in the email, or submit it via the BlueFish button in Outlook for investigation. If you don't have the BlueFish button or are using a mobile device, forward the email as an attachment to ***@***.******@***.***?subject=Report%20a%20Suspicious%20Email>

________________________________ At the moment Platforma can only start from FASTQ files but in a few weeks we will add a feature to upload repertoires from tsv tables. — Reply to this email directly, view it on GitHub<#1910 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BH7E6ZKHXC6GXNOOBZVINY32TC66RAVCNFSM6AAAAABYEPLTX6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTENBRHEZDENI>. You are receiving this because you authored the thread.Message ID: ***@***.***> Please consider the environment before printing this e-mail Cleveland Clinic is a nonprofit, multispecialty academic medical center that's recognized in the U.S. and throughout the world for its expertise and care. Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you.

[mizraelson]

Hi, write now we are still working on the documentation for Platforma. Feel free to reach out to our Platforma team on https://community.platforma.bio/ and ask for assistance.

[singhd6]

Sure, Thank you. Divya

…

________________________________ From: mizraelson ***@***.***> Sent: Wednesday, March 12, 2025 12:58 PM To: milaboratory/mixcr ***@***.***> Cc: Singh, Divya Jyoti ***@***.***>; Author ***@***.***> Subject: [EXT] Re: [milaboratory/mixcr] Error during creating the SHMtree plot (Discussion #1910) PROCEED WITH CAUTION: Slow down and pay close attention to emails sent from outside the organization. If you receive an unsolicited email from an unknown sender or are suspicious of the tone, style, vocabulary or urgency of the email message, never click links or open attachments within it. When in doubt, you should either delete the email, verify its authenticity by contacting the sender using an alternative method not listed in the email, or submit it via the BlueFish button in Outlook for investigation. If you don't have the BlueFish button or are using a mobile device, forward the email as an attachment to ***@***.******@***.***?subject=Report%20a%20Suspicious%20Email>

________________________________ Hi, write now we are still working on the documentation for Platforma. Feel free to reach out to our Platforma team on https://community.platforma.bio/<https://community.platforma.bio/> and ask for assistance. — Reply to this email directly, view it on GitHub<#1910 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BH7E6ZLZMPWHA6OQVHZXFXT2UBRUBAVCNFSM6AAAAABYEPLTX6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTENBXG43TMNY>. You are receiving this because you authored the thread.Message ID: ***@***.***> Please consider the environment before printing this e-mail Cleveland Clinic is a nonprofit, multispecialty academic medical center that's recognized in the U.S. and throughout the world for its expertise and care. Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you.