Help with clonotype analysis of BCR cDNA library

[liyunjian]

I always see a high percentage of Unassigned alignments in clonotype assembly in the output of MIXCR.
The output clones are around 8000. I want to ask if this is normal.
This is a BCR cDNA library sequenced by Illumina Novaseq6000 SP PE250。

The command I used is as follows:
mixcr analyze generic-amplicon-with-umi ./oneUMI/JLW2KL_R1A.fastq ./oneUMI/JLW2KL_R2A.fastq ./oneUMI/JLW2KL
-Xmx50g
-Massemble.cloneAssemblerParameters.addReadsCountOnClustering=true
--species hsa
--rna
--tag-pattern "^(UMI:N{8})(R1:)^(R2:)"
--floating-left-alignment-boundary
--floating-right-alignment-boundary J
--assemble-clonotypes-by "VDJRegion"
--impute-germline-on-export
--threads 32

Here is the report I output:
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Analysis date: Sun May 26 16:14:09 CST 2024
Input file(s): ./oneUMI/JLW2H_R1A.fastq,./oneUMI/JLW2H_R2A.fastq
Output file(s): ./oneUMI/JLW2H/JLW2H.vdjca
Version: 4.6.0; built=Sun Dec 10 03:48:42 CST 2023; rev=c9fafa41fe; lib=repseqio.v4.0
Command line arguments: align --report ./oneUMI/JLW2H/JLW2H.align.report.txt --json-report ./oneUMI/JLW2H/JLW2H.align.report.json --threads 32 --preset generic-amplicon-with-umi --save-output-file-names ./oneUMI/JLW2H/JLW2H.align.list --floating-left-alignment-boundary --floating-right-alignment-boundary J --rna --species hsa --tag-pattern ^(UMI:N{8})(R1:)^(R2:) --assemble-clonotypes-by VDJRegion --impute-germline-on-export -M assemble.cloneAssemblerParameters.addReadsCountOnClustering=true ./oneUMI/JLW2H_R1A.fastq ./oneUMI/JLW2H_R2A.fastq ./oneUMI/JLW2H/JLW2H.vdjca
Analysis time: 3.47m
Total sequencing reads: 1231835
Successfully aligned reads: 1200236 (97.43%)
Coverage (percent of successfully aligned):
CDR3: 1189531 (99.11%)
FR3_TO_FR4: 1117808 (93.13%)
CDR2_TO_FR4: 1117778 (93.13%)
FR2_TO_FR4: 1116756 (93.04%)
CDR1_TO_FR4: 1116359 (93.01%)
VDJRegion: 1114776 (92.88%)
Alignment failed: no hits (not TCR/IG?): 3885 (0.32%)
Alignment failed after alignment-aided overlap: 12 (0%)
Alignment failed: absence of V hits: 6 (0%)
Alignment failed: absence of J hits: 26339 (2.14%)
Alignment failed: no target with both V and J alignments: 1357 (0.11%)
Overlapped: 1223011 (99.28%)
Overlapped and aligned: 1199080 (97.34%)
Overlapped and not aligned: 23931 (1.94%)
Alignment-aided overlaps, percent of overlapped and aligned: 707 (0.06%)
No CDR3 parts alignments, percent of successfully aligned: 105 (0.01%)
Partial aligned reads, percent of successfully aligned: 10600 (0.88%)
V gene chimeras: 386 (0.03%)
Paired-end alignment conflicts eliminated: 324 (0.03%)
Realigned with forced non-floating bound: 19062 (1.55%)
Realigned with forced non-floating right bound in left read: 311 (0.03%)
Realigned with forced non-floating left bound in right read: 311 (0.03%)
IGH chains: 1200071 (99.99%)
IGH non-functional: 17462 (1.46%)
IGK chains: 53 (0%)
IGK non-functional: 0 (0%)
IGL chains: 112 (0.01%)
IGL non-functional: 4 (3.57%)
Trimming report:
R1 reads trimmed left: 1 (0%)
R1 reads trimmed right: 15 (0%)
Average R1 nucleotides trimmed left: 2.435391103516299E-6
Average R1 nucleotides trimmed right: 6.859684941570908E-4
R2 reads trimmed left: 230 (0.02%)
R2 reads trimmed right: 89 (0.01%)
Average R2 nucleotides trimmed left: 3.888507795281024E-4
Average R2 nucleotides trimmed right: 0.0030101434039461455
Tag parsing report:
Execution time: 0ns
Total reads: 1231835
Matched reads: 1231835 (100%)
Projection +R1 +R2: 1231835 (100%)
For variant 0:
For projection +R1 +R2:
UMI:Left position: 0
R1:Left position: 8
UMI:Right position: 8
R1:Right position:
186249: + 4694 (0.38%) = 4694 (0.38%)
250: + 1227141 (99.62%) = 1231835 (100%)
R2:Left position: 0
Variants: 0
Cost: 0
UMI length: 8
R1 length:
178241: + 4694 (0.38%) = 4694 (0.38%)
242: + 1227141 (99.62%) = 1231835 (100%)
R2 length: 250
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Analysis date: Sun May 26 16:19:13 CST 2024
Input file(s): ./oneUMI/JLW2H/JLW2H.vdjca
Output file(s): ./oneUMI/JLW2H/JLW2H.refined.vdjca
Version: 4.6.0; built=Sun Dec 10 03:48:42 CST 2023; rev=c9fafa41fe; lib=repseqio.v4.0
Command line arguments: refineTagsAndSort --report ./oneUMI/JLW2H/JLW2H.refine.report.txt --json-report ./oneUMI/JLW2H/JLW2H.refine.report.json ./oneUMI/JLW2H/JLW2H.vdjca ./oneUMI/JLW2H/JLW2H.refined.vdjca
Analysis time: 1.59m
Time spent in correction: 1.12m
Number of input records: 1200236
Number of output records: 1185174 (98.75%)
UMI correction report:
UMI input diversity: 41676
UMI output diversity: 33572 (80.55%)
UMI input reads: 1200236
UMI output reads: 1200234 (100%)
UMI mean reads per tag: 28.8
UMI input core diversity: 39869 (95.66%)
UMI input core reads: 1198429 (99.85%)
UMI directly corrected diversity: 8102 (19.44%)
UMI directly corrected reads: 8953 (0.75%)
UMI diversity filtered by tag quality: 2 (0%)
UMI reads filtered by tag quality: 2 (0%)
UMI diversity filtered by whitelist: 0 (0%)
UMI recursively corrected: 724
Filter report:
Number of groups: 33572
Number of groups accepted: 28514 (84.93%)
Total records weight: 1200234
Records weight accepted: 1185174 (98.75%)
Operator #0:
Effective threshold: 6.0
Nested thresholds:
#0: 6
#1: 26
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Analysis date: Sun May 26 16:19:13 CST 2024
Input file(s): ./oneUMI/JLW2H/JLW2H.refined.vdjca
Output file(s): ./oneUMI/JLW2H/JLW2H.clns
Version: 4.6.0; built=Sun Dec 10 03:48:42 CST 2023; rev=c9fafa41fe; lib=repseqio.v4.0
Command line arguments: assemble --report ./oneUMI/JLW2H/JLW2H.assemble.report.txt --json-report ./oneUMI/JLW2H/JLW2H.assemble.report.json ./oneUMI/JLW2H/JLW2H.refined.vdjca ./oneUMI/JLW2H/JLW2H.clns
Analysis time: 42.54m
Final clonotype count: 7356
Reads used in clonotypes, percent of total: 340261 (27.62%)
Average number of reads per clonotype: 46.26
Reads dropped due to the lack of a clone sequence, percent of total: 84133 (6.83%)
Reads dropped due to a too short clonal sequence, percent of total: 0 (0%)
Reads dropped due to low quality, percent of total: 0 (0%)
Reads dropped due to failed mapping, percent of total: 36814 (2.99%)
Reads dropped with low quality clones, percent of total: 5178 (0.42%)
Aligned reads processed: 382253
Reads used in clonotypes before clustering, percent of total: 340261 (27.62%)
Number of reads used as a core, percent of used: 338087 (99.36%)
Mapped low quality reads, percent of used: 2174 (0.64%)
Reads clustered in PCR error correction, percent of used: 1378 (0.4%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 0 (0%)
Clonotypes dropped as low quality: 298
Clonotypes eliminated by PCR error correction: 61
Clonotypes pre-clustered due to the similar VJC-lists: 0
Clones dropped in post filtering: 0 (0%)
Reads dropped in post filtering: 0.0 (0%)
Alignments filtered by tag prefix: 0 (0%)
IGH chains: 7356 (100%)
IGH non-functional: 130 (1.77%)
Pre-clone assembler report:
Number of input groups: 28514
Number of input groups with no assembling feature: 38
Number of input alignments: 1185174
Number of alignments with assembling feature: 1101041 (92.9%)
Number of output pre-clones: 20474
Number of pre-clonotypes per group:
0: + 9494 (33.34%) = 9494 (33.34%)
1: + 17525 (61.54%) = 27019 (94.88%)
2: + 1422 (4.99%) = 28441 (99.88%)
3: + 35 (0.12%) = 28476 (100%)
Number of assembling feature sequences in groups with zero pre-clonotypes: 467190
Number of dropped pre-clones by tag suffix conflict: 0
Number of dropped alignments by tag suffix conflict: 0
Number of core alignments: 379286 (32%)
Discarded core alignments: 721755 (190.29%)
Empirically assigned alignments: 2967 (0.25%)
Empirical assignment conflicts: 86 (0.01%)
Tag+VJ-gene empirically assigned alignments: 3053 (0.26%)
VJ-gene empirically assigned alignments: 0 (0%)
Tag empirically assigned alignments: 0 (0%)
Number of ambiguous groups: 1457
Number of ambiguous V-genes: 321
Number of ambiguous J-genes: 595
Number of ambiguous tag+V/J-gene combinations: 916
Ignored non-productive alignments: 0 (0%)
Unassigned alignments: 802470 (67.71%)
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[mizraelson]

One of the major reasons for that could be a short UMI sequence.
0: + 9494 (33.34%) = 9494 (33.34%)
For 9494 UMIs, no consensus was assembled, possibly because there were multiple clones marked with the same UMI. For example, if a UMI is covered by 10 reads, but each read has a different CDR3, no consensus will be assembled.

[liyunjian]

thank you for your reply.