QIAgen Targeted library

[GabyBG]

Hello,

Thank you so much for mixer, it has proven to be a very valuable package for my Immune repertoire analysis.
I am currently working with QIAseq Targeted RNA Panel TCR Library Kit, I tried running mixcr version 4.6 👍

mixcr analyze qiagen-human-rna-tcr-umi-qiaseq fastq.R1.fastq.gz  fastq.R2.fastq.gz

Very few reads aligned and it could be because the configuration of the library might be a little different. According to the vendor the library has phasing on both ends.
Are there any plans to dd the targetedRNA Panel TCR library kit configuration? or is it something I missed in the documentation? When comparing the results from the pipeline described above vs the Qiagen analysis tool, the results are very different.

Thank you so much for your help!

[mizraelson]

Hi,
Can you share the align report here?

[GabyBG]

thank you for your promt response!
here is the align report

Analysis date: Wed Jul 31 20:16:21 UTC 2024
Input file(s): SM313_01_S1_R1_001.fastq.gz,SM313_01_S1_R2_001.fastq.gz
Output file(s): results/alignments.vdjca
Version: 4.6.0; built=Sat Dec 09 19:48:42 UTC 2023; rev=c9fafa41fe; lib=repseqio.v4.0
Command line arguments: align -f --report results/align.report.txt --json-report results/align.report.json --preset qiagen-human-rna-tcr-umi-qiaseq --save-output-file-name
s results/align.list SM313_01_S1_R1_001.fastq.gz SM313_01_S1_R2_001.fastq.gz results/alignments.vdjca
Analysis time: 3.16m
Total sequencing reads: 17390174
Successfully aligned reads: 1768 (0.01%)
Coverage (percent of successfully aligned):
  CDR3: 1759 (99.49%)
  FR3_TO_FR4: 1686 (95.36%)
  CDR2_TO_FR4: 1465 (82.86%)
  FR2_TO_FR4: 864 (48.87%)
  CDR1_TO_FR4: 856 (48.42%)
  VDJRegion: 773 (43.72%)
Alignment failed: no hits (not TCR/IG?): 20 (0%)
Alignment failed after alignment-aided overlap: 217 (0%)
Alignment failed: absence of V hits: 2 (0%)
Alignment failed: absence of J hits: 10 (0%)
Alignment failed: no target with both V and J alignments: 6 (0%)
Alignment failed: absent barcode: 17388151 (99.99%)
Overlapped: 908 (0.01%)
Overlapped and aligned: 883 (0.01%)
Overlapped and not aligned: 25 (0%)
Alignment-aided overlaps, percent of overlapped and aligned: 0 (0%)
Partial aligned reads, percent of successfully aligned: 9 (0.51%)
V gene chimeras: 2 (0%)
Paired-end alignment conflicts eliminated: 2 (0%)
Realigned with forced non-floating bound: 1115 (0.01%)
Realigned with forced non-floating right bound in left read: 59 (0%)
Realigned with forced non-floating left bound in right read: 59 (0%)
TRA chains: 1025 (57.98%)
TRA non-functional: 44 (4.29%)
TRB chains: 697 (39.42%)
TRB non-functional: 8 (1.15%)
TRD chains: 1 (0.06%)
TRD non-functional: 0 (0%)
TRG chains: 45 (2.55%)
TRG non-functional: 19 (42.22%)
Trimming report:
  R1 reads trimmed left: 8308 (0.05%)
  R1 reads trimmed right: 2022 (0.01%)
  Average R1 nucleotides trimmed left: 0.0020616239952515715
  Average R1 nucleotides trimmed right: 0.00126784240341701
  R2 reads trimmed left: 22013 (0.13%)
  R2 reads trimmed right: 425 (0%)
  Average R2 nucleotides trimmed left: 0.01516896840710162
  Average R2 nucleotides trimmed right: 0.0015713471297066953
Tag parsing report:
  Execution time: 0ns
  Total reads: 17390174
  Matched reads: 2023 (0.01%)
  Projection +R1 +R2: 2023 (0.01%)
  For variant 0:
    For projection +R1 +R2:
      R1:Left position:
        21~25: + 455 (22.49%) = 455 (22.49%)
        26~27: + 344 (17%) = 799 (39.5%)
        28: + 504 (24.91%) = 1303 (64.41%)
        29~30: + 330 (16.31%) = 1633 (80.72%)
        31~37: + 390 (19.28%) = 2023 (100%)
      R1:Right position:
        109~249: + 273 (13.49%) = 273 (13.49%)
        250: + 472 (23.33%) = 745 (36.83%)
        251: + 1278 (63.17%) = 2023 (100%)
      UMI:Left position: 0
      UMI:Right position: 12
      R2:Left position: 24
      Variants: 0
      Cost:
        0: + 1673 (82.7%) = 1673 (82.7%)
        5: + 350 (17.3%) = 2023 (100%)
      R1 length:
        80~219: + 357 (17.65%) = 357 (17.65%)
        220~221: + 363 (17.94%) = 720 (35.59%)
        222: + 233 (11.52%) = 953 (47.11%)
        223: + 371 (18.34%) = 1324 (65.45%)
        224~225: + 342 (16.91%) = 1666 (82.35%)
        226~230: + 357 (17.65%) = 2023 (100%)
      UMI length: 12
      R2 length: 227
======================================

[mizraelson]

Do you know the common sequence and the phasing size on each end? From the report I see that the pattern hasn't been identified meaning the primers were not found.
As a workaround you can try and add:

mixcr analyze qiagen-human-rna-tcr-umi-qiaseq \
--tag-pattern "^(R1:*)\^(UMI:N{18})N{10}(R2:*)" \
fastq.R1.fastq.gz  \
fastq.R2.fastq.gz \
output