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I ran the same pipeline on a shallow and deep sequencing file of the same sample. Based on exportAlignments output, there is a pre-clone that is counted as a clonotype in the shallow analysis (cloneIdWithMappingType=2:core), but then it is dropped in the deep analysis (cloneIdWithMappingType=-1:dropped). I have checked that the reads belonging to the clone exists in both sequencing files.
Is there a way to get more details on why the clonotype group was dropped? What parameters could be causing a greater percentage of Discarded core alignments? Are there cutoffs that are determined relative to the whole sample?
Assemble report for shallow sample:
Version: 4.2.0; built=Thu Jan 26 11:40:26 PST 2023; rev=b0f194e0c6; lib=repseqio.v2.1
Command line arguments: assemble --report ../../data/2023-03-08_p53_nanopore/mixcr_24h_demult_0.1_trim/E1.assemble.report.txt --json-report ../../data/2023-03-08_p53_nanopore/mixcr_24h_demult_0.1_trim/E1.assemble.report.json ../../data/2023-03-08_p53_nanopore/mixcr_24h_demult_0.1_trim/E1.refined.vdjca ../../data/2023-03-08_p53_nanopore/mixcr_24h_demult_0.1_trim/E1.clns
Analysis time: 11.38s
Number of input groups: 3
Number of groups with no assembling feature: 0
Number of input alignments: 56608
Number of output pre-clonotypes: 9
Number alignments with assembling feature: 54818 (96.84%)
Number of clonotypes per group: 3
Number of assembling feature sequences in groups with zero pre-clonotypes: 0
Number of core alignments: 50468 (89.15%)
Discarded core alignments: 4350 (8.62%)
Empirically assigned alignments: 36 (0.06%)
Empirical assignment conflicts: 0 (0%)
UMI+VJ-gene empirically assigned alignments: 36 (0.06%)
VJ-gene empirically assigned alignments: 0 (0%)
UMI empirically assigned alignments: 0 (0%)
Number of ambiguous UMIs: 23
Number of ambiguous UMI+V/J-gene combinations: 0
Unassigned alignments: 6104 (10.78%)
Final clonotype count: 8
Average number of reads per clonotype: 6313
Reads used in clonotypes, percent of total: 50504 (54.65%)
Reads used in clonotypes before clustering, percent of total: 50504 (54.65%)
Number of reads used as a core, percent of used: 50504 (100%)
Mapped low quality reads, percent of used: 0 (0%)
Reads clustered in PCR error correction, percent of used: 0 (0%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 0 (0%)
Reads dropped due to the lack of a clone sequence, percent of total: 1790 (1.94%)
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: 0 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Clonotypes eliminated by PCR error correction: 0
Clonotypes dropped as low quality: 0
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%)
TRA chains: 6 (75%)
TRA non-functional: 0 (0%)
TRB chains: 2 (25%)
TRB non-functional: 0 (0%)
Assemble report for deep sample:
Version: 4.2.0; built=Thu Jan 26 11:40:26 PST 2023; rev=b0f194e0c6; lib=repseqio.v2.1
Command line arguments: assemble --report ../../data/2023-03-08_p53_nanopore/mixcr_raw_demult_0.1_trim/E1.assemble.report.txt --json-report ../../data/2023-03-08_p53_nanopore/mixcr_raw_demult_0.1_trim/E1.assemble.report.json ../../data/2023-03-08_p53_nanopore/mixcr_raw_demult_0.1_trim/E1.refined.vdjca ../../data/2023-03-08_p53_nanopore/mixcr_raw_demult_0.1_trim/E1.clns
Analysis time: 19.62s
Number of input groups: 3
Number of groups with no assembling feature: 0
Number of input alignments: 90062
Number of output pre-clonotypes: 7
Number alignments with assembling feature: 87038 (96.64%)
Number of clonotypes per group:
1: + 1 (33.33%) = 1 (33.33%)
3: + 2 (66.67%) = 3 (100%)
Number of assembling feature sequences in groups with zero pre-clonotypes: 0
Number of core alignments: 59714 (66.3%)
Discarded core alignments: 27324 (45.76%)
Empirically assigned alignments: 54 (0.06%)
Empirical assignment conflicts: 0 (0%)
UMI+VJ-gene empirically assigned alignments: 54 (0.06%)
VJ-gene empirically assigned alignments: 0 (0%)
UMI empirically assigned alignments: 0 (0%)
Number of ambiguous UMIs: 19
Number of ambiguous UMI+V/J-gene combinations: 0
Unassigned alignments: 30294 (33.64%)
Final clonotype count: 6
Average number of reads per clonotype: 9961.33
Reads used in clonotypes, percent of total: 59768 (40.46%)
Reads used in clonotypes before clustering, percent of total: 59768 (40.46%)
Number of reads used as a core, percent of used: 59768 (100%)
Mapped low quality reads, percent of used: 0 (0%)
Reads clustered in PCR error correction, percent of used: 0 (0%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 0 (0%)
Reads dropped due to the lack of a clone sequence, percent of total: 3024 (2.05%)
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: 0 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Clonotypes eliminated by PCR error correction: 0
Clonotypes dropped as low quality: 0
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%)
TRA chains: 5 (83.33%)
TRA non-functional: 0 (0%)
TRB chains: 1 (16.67%)
TRB non-functional: 0 (0%)
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I ran the same pipeline on a shallow and deep sequencing file of the same sample. Based on
exportAlignments
output, there is a pre-clone that is counted as a clonotype in the shallow analysis (cloneIdWithMappingType=2:core
), but then it is dropped in the deep analysis (cloneIdWithMappingType=-1:dropped
). I have checked that the reads belonging to the clone exists in both sequencing files.Is there a way to get more details on why the clonotype group was dropped? What parameters could be causing a greater percentage of
Discarded core alignments
? Are there cutoffs that are determined relative to the whole sample?Assemble report for shallow sample:
Assemble report for deep sample:
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