low Successfully aligned reads in bulk rna-seq data

[Mengzhensw]

Checklist before submitting the issue:

• The issue is strongly related to the MiXCR software
• The issue can be reproduced with the most recent version of MiXCR
• There is no answer to the question in the official documentation and there is no duplicate issue in the bug tracker
• Inspection of raw alignments with exportAlignmentsPretty shows that data has the expected architecture, and sample preparation artefacts are not the reason of the problem (if this is the matter of the issue)

Expected Result

Hi, I really appreciate this great software. I am using bulk RNA-seq data from whole blood to perform MiXCR analysis, but I am getting a very low successfully aligned read rate—around 0–0.03%. The library was prepared using total RNA. I understand that the alignment rate should be low since the sequencing data is not specifically targeted for TCR or BCR. However, I would like to confirm whether this is the expected alignment rate and if I can proceed with downstream analysis.

Exact MiXCR commands

mixcr analyze rna-seq --species hsa P0097/P0097_64_S62_L002_R1_001.fastq.gz P0097/P0097_64_S62_L002_R2_001.fastq.gz P0097/mixcr_res/P0097_64

MiXCR report files

input file(s): P0097/P0097_64_S62_L002_R1_001.fastq.gz,P0097/P0097_64_S62_L002_R2_001.fastq.gz
Output file(s): P0097/mixcr_res/P0097_64.alignments.vdjca
Version: 4.7.0; built=Wed Aug 07 21:19:48 CEST 2024; rev=976ba14139; lib=repseqio.v5.1
Command line arguments: align --report P0097/mixcr_res/P0097_64.align.report.txt --json-report P0097/mixcr_res/P0097_64.align.report.json --preset rna-seq --save-output-file-names P0097/mixcr_res/P0097_64.align.list.tsv --species hsa P0097/P0097_64_S62_L002_R1_001.fastq.gz P0097/P0097_64_S62_L002_R2_001.fastq.gz P0097/mixcr_res/P0097_64.alignments.vdjca
Analysis time: 4.27m
Total sequencing reads: 43463054
Successfully aligned reads: 9628 (0.02%)
Coverage (percent of successfully aligned):
CDR3: 2927 (30.4%)
FR3_TO_FR4: 11 (0.11%)
CDR2_TO_FR4: 3 (0.03%)
FR2_TO_FR4: 1 (0.01%)
CDR1_TO_FR4: 1 (0.01%)
VDJRegion: 0 (0%)
Alignment failed: no hits (not TCR/IG?): 41689342 (95.92%)
Alignment failed after alignment-aided overlap: 2 (0%)
Alignment failed: no CDR3 parts: 39365 (0.09%)
Alignment failed: low total score: 1724717 (3.97%)
Overlapped: 22409922 (51.56%)
Overlapped and aligned: 4121 (0.01%)
Overlapped and not aligned: 22405801 (51.55%)
Alignment-aided overlaps, percent of overlapped and aligned: 337 (8.18%)
No CDR3 parts alignments, percent of successfully aligned: 184 (1.91%)
Partial aligned reads, percent of successfully aligned: 6517 (67.69%)
V gene chimeras: 44 (0%)
J gene chimeras: 21 (0%)
Paired-end alignment conflicts eliminated: 8 (0%)
Realigned with forced non-floating bound: 42106938 (96.88%)
Realigned with forced non-floating right bound in left read: 4700 (0.01%)
Realigned with forced non-floating left bound in right read: 4700 (0.01%)
TRA chains: 1344 (13.96%)
TRA non-functional: 30 (2.23%)
TRB chains: 915 (9.5%)
TRB non-functional: 14 (1.53%)
TRD chains: 76 (0.79%)
TRD non-functional: 1 (1.32%)
TRG chains: 300 (3.12%)
TRG non-functional: 12 (4%)
IGH chains: 3292 (34.19%)
IGH non-functional: 21 (0.64%)
TRAD chains: 95 (0.99%)
TRAD non-functional: 0 (0%)
IGK chains: 1673 (17.38%)
IGK non-functional: 13 (0.78%)
IGL chains: 1933 (20.08%)
IGL non-functional: 23 (1.19%)
Trimming report:
R1 reads trimmed left: 1666 (0%)
R1 reads trimmed right: 8786 (0.02%)
Average R1 nucleotides trimmed left: 2.566317590107681E-4
Average R1 nucleotides trimmed right: 0.001156591527139349
R2 reads trimmed left: 1293 (0%)
R2 reads trimmed right: 8713 (0.02%)
Average R2 nucleotides trimmed left: 3.1495715878594266E-4
Average R2 nucleotides trimmed right: 0.0017106483129326346

[mizraelson]

Hi,
The command is correct. The numbers you get are expected and may vary from sample to sample.