Request for Assistance with MiXCR Analysis of TCR Repertoire Data

[PpGrApee]

Hello！
I encountered some issues while running the code and would like to seek your help here.
I'm analyzing TCR sequencing data from 28 healthy mice in the paper titled "T-cell receptor repertoires share a restricted set of public and abundant CDR3 sequences that are associated with self-related immunity".
I first preprocessed the raw data by removing the first 3 bases (barcode sequences). Then i used the generic-amplicon preset for initial analysis, but got low alignment rate (~6% of reads aligned). So i am trying to perform step-by-step analysis, but i encountered errors in my pipeline.
Could you advise on the proper MiXCR analysis approach for this dataset? Below are my code attempts and corresponding error messages:
1. First Generation Code:
mixcr -Xmx70G analyze generic-amplicon
--species mmu
--rna
--floating-left-alignment-boundary --floating-right-alignment-boundary J
--assemble-clonotypes-by CDR3 --verbose
${name}.fastq.gz
${analy}/${name}
2. Second Generation Code:
mixcr -Xmx70G align
--report ${analy}/${name}.align.report.txt
--json-report ${analy}/${name}.align.report.json
--preset generic-amplicon
--save-output-file-names ${analy}/${name}.align.list.tsv
--species mmu
--rna
--assemble-clonotypes-by CDR3
--library imgt.202312-3.sv8
--floating-left-alignment-boundary
--floating-right-alignment-boundary J
-OvParameters.parameters.minAlignmentLength=11
-OjParameters.parameters.minAlignmentLength=9
${name}.fastq.gz
${analy}/${name}.alignments.vdjca \

ERROR info:
picocli.CommandLine$ExecutionException: Error while running command align java.lang.RuntimeException: Failed to override some parameter.

3. Here is the method that this paper build the TCR library:
Libraries were prepared and pre-processed as published (Ndifon et al. 2012). Briefly, we extracted total RNA from T cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA was then reverse transcribed using SuperScript II reverse transcriptase (RT) enzyme (Invitrogen, La Jolla, CA). The primer for the RT reaction was a TCR Cβ-specific primer linked to the 3'-end Illumina sequencing adapter. The resulting cDNA was then amplified using PCR (Phusion; Finnzymes) with a Cβ-3’adp primer and Vβ-specific 5’ primers. Each Vβ-specific primer was anchored to a restriction site sequence for the ACUI restriction enzyme. PCR products were then cleaned using QIAquick PCR purification kit (Qiagen, Hilden, Germany), followed by enzymatic digestion with ACUI enzyme (New England BioLabs, Ipswich, MA). Then, the 5’Illumina adaptor (dsDNA, with NN overhang) were ligated (T4 ligase; Fermentas, Vilnius, Lithuania). The adaptors also contained 3-nucleotide long tags for multiplexing of samples to the same Illumina sequencing run. A second round of PCR amplification was performed, using universal primers for the 5’ and 3’ Illumina adapters. Final PCR products were run on a 2% agarose gel, cut at the desired length (~250bp), and purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) to produce the final library. The libraries were sequenced using Genome Analyzer II or HiSeq2000 (Illumina).

Thank you in advance for your help!!!!!!

[mizraelson]

Hi, could you share your initial alignment report? If the reads didn’t align, that usually indicates a major issue, and running the step-by-step process likely won’t help much.

[PpGrApee]

Hi, could you share your initial alignment report? If the reads didn’t align, that usually indicates a major issue, and running the step-by-step process likely won’t help much.

Of course, The attachment is the alignment report for one of the samples. Thank you for your help![

SRR1339467.align.report.txt

](url)