Low alignment rate when using fastq generated from bamtofastq

[malonzm1]

Hi,

I had to use cellranger's bamtofastq function for some bam files. Then I used the resulting fastq files for clonotype detection with mixcr. I noticed that the alignment rate was very low (from less than 1% to around 3%). Any thoughts?

Thanks and good day.

[mizraelson]

Its hard to say without knowing the wet-lab details. What cells were used for the sample preparation ? If it is a 10x data - what kit was used? 5'10x VDJ? What command did you use for MiXCR analysis? Can you share the report files generated by MiXCR?

[malonzm1]

Thanks. I analyzed various studies. E.g. GSE160097 which used PBMCs and 5' 10x VDJ, GSE138669 which used skin and 3' 10x V2, GSE151177 which used skin and 3' 10x, GSE179640 which used endometrium and 3' 10x V3, and more.

I used the following command:

mixcr analyze %s -s hsa --rna --threads 4 --force-overwrite %s %s %s/%s'%(preset,infile1,infile2,outdir,srr)

One example of report file.

>>>>>>>>>>>>>>>>>>>>>>> mixcr align <<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr align --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.align.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.align.report.json --threads 4 --preset 10x-sc-5gex --rna --species hsa /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE151177/SRR11848679/fastq/bamtofastq_R1.fastq.gz /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE151177/SRR11848679/fastq/bamtofastq_R2.fastq.gz /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.vdjca
The following tags and their roles will be associated with each output alignment:
  Payload tags: R2
  Cell tags: CELL(SQ)
  Molecule tags: UMI(SQ)
Alignment: 0%
Alignment: 1.2%  ETA: 02:41:51
Alignment: 2.8%  ETA: 02:07:00
Alignment: 4.6%  ETA: 01:45:30
Alignment: 6.4%  ETA: 01:44:05
Alignment: 7.7%  ETA: 02:19:04
Alignment: 9.1%  ETA: 02:11:44
Alignment: 10.3%  ETA: 02:21:23
Alignment: 12.1%  ETA: 01:38:10
Alignment: 13.9%  ETA: 01:35:46
Alignment: 15.5%  ETA: 01:45:00
Alignment: 16.9%  ETA: 02:00:32
Alignment: 18.3%  ETA: 01:53:51
Alignment: 20.3%  ETA: 01:22:01
Alignment: 22.1%  ETA: 01:27:29
Alignment: 23.7%  ETA: 01:35:09
Alignment: 25.3%  ETA: 01:34:47
Alignment: 26.5%  ETA: 01:53:54
Alignment: 27.9%  ETA: 01:42:47
Alignment: 29.4%  ETA: 01:37:20
Alignment: 30.8%  ETA: 01:36:34
Alignment: 32.3%  ETA: 01:34:30
Alignment: 33.8%  ETA: 01:29:04
Alignment: 35.2%  ETA: 01:31:14
Alignment: 36.8%  ETA: 01:15:21
Alignment: 38.4%  ETA: 01:17:15
Alignment: 39.8%  ETA: 01:26:54
Alignment: 41.2%  ETA: 01:27:38
Alignment: 42.7%  ETA: 01:17:15
Alignment: 44.8%  ETA: 00:52:31
Alignment: 46.4%  ETA: 01:05:32
Alignment: 48%  ETA: 01:06:38
Alignment: 49.3%  ETA: 01:18:15
Alignment: 50.5%  ETA: 01:21:56
Alignment: 51.5%  ETA: 01:37:06
Alignment: 52.6%  ETA: 01:25:57
Alignment: 53.8%  ETA: 01:18:01
Alignment: 54.9%  ETA: 01:19:26
Alignment: 56%  ETA: 01:15:53
Alignment: 57.2%  ETA: 01:16:56
Alignment: 58.3%  ETA: 01:13:50
Alignment: 59.5%  ETA: 01:09:53
Alignment: 60.5%  ETA: 01:13:45
Alignment: 61.7%  ETA: 01:04:27
Alignment: 62.8%  ETA: 01:06:20
Alignment: 64%  ETA: 01:01:49
Alignment: 65.1%  ETA: 01:01:50
Alignment: 66.3%  ETA: 00:58:55
Alignment: 67.4%  ETA: 00:57:51
Alignment: 68.7%  ETA: 00:48:29
Alignment: 70.1%  ETA: 00:41:31
Alignment: 71.4%  ETA: 00:45:39
Alignment: 72.6%  ETA: 00:43:34
Alignment: 73.9%  ETA: 00:42:48
Alignment: 75.1%  ETA: 00:41:15
Alignment: 76.1%  ETA: 00:45:01
Alignment: 77.1%  ETA: 00:46:30
Alignment: 78.2%  ETA: 00:39:42
Alignment: 79.3%  ETA: 00:36:32
Alignment: 80.5%  ETA: 00:35:06
Alignment: 81.6%  ETA: 00:32:52
Alignment: 82.6%  ETA: 00:32:44
Alignment: 83.7%  ETA: 00:29:12
Alignment: 84.9%  ETA: 00:27:19
Alignment: 85.9%  ETA: 00:26:18
Alignment: 87%  ETA: 00:23:41
Alignment: 88.1%  ETA: 00:22:07
Alignment: 89.3%  ETA: 00:18:18
Alignment: 90.4%  ETA: 00:17:48
Alignment: 91.5%  ETA: 00:15:05
Alignment: 92.6%  ETA: 00:13:32
Alignment: 93.9%  ETA: 00:09:21
Alignment: 95.3%  ETA: 00:06:46
Alignment: 96.5%  ETA: 00:05:49
Alignment: 97.7%  ETA: 00:03:43
Alignment: 98.9%  ETA: 00:01:53
====================== report: align ======================
Analysis time: 151.93m
Total sequencing reads: 504461402
Successfully aligned reads: 7559118 (1.5%)
Coverage (percent of successfully aligned):
  CDR3: 0%
  FR3_TO_FR4: 0%
  CDR2_TO_FR4: 0%
  FR2_TO_FR4: 0%
  CDR1_TO_FR4: 0%
  VDJRegion: 0%
Alignment failed: no hits (not TCR/IG?): 482078562 (95.56%)
Alignment failed: low total score: 14823722 (2.94%)
Overlapped: 0 (0%)
Overlapped and aligned: 0 (0%)
Overlapped and not aligned: 0 (0%)
Alignment-aided overlaps, percent of overlapped and aligned: 0 (NaN%)
No CDR3 parts alignments, percent of successfully aligned: 7480597 (98.96%)
Partial aligned reads, percent of successfully aligned: 78278 (1.04%)
Realigned with forced non-floating bound: 0 (0%)
Realigned with forced non-floating right bound in left read: 0 (0%)
Realigned with forced non-floating left bound in right read: 0 (0%)
TRA chains: 12436 (0.16%)
TRA non-functional: 31 (0.25%)
TRB chains: 827316 (10.94%)
TRB non-functional: 2 (0%)
TRD chains: 6104 (0.08%)
TRD non-functional: 0 (0%)
TRG chains: 3791620 (50.16%)
TRG non-functional: 29 (0%)
IGH chains: 637012 (8.43%)
IGH non-functional: 0 (0%)
TRAD chains: 989930 (13.1%)
TRAD non-functional: 0 (0%)
IGK chains: 423117 (5.6%)
IGK non-functional: 0 (0%)
IGL chains: 871583 (11.53%)
IGL non-functional: 0 (0%)
Trimming report:
  R1 reads trimmed left: 330540 (0.07%)
  R1 reads trimmed right: 707876 (0.14%)
  Average R1 nucleotides trimmed left: 0.01378815697776616
  Average R1 nucleotides trimmed right: 0.04743556177961064
Tag parsing report:
  Execution time: 0ns
  Total reads: 504461402
  Matched reads: 504461402 (100%)
  Projection +R1 +R2: 504461402 (100%)
  For variant 0:
    For projection +R1 +R2:
      CELL:Left position: 0
      UMI:Left position: 16
      CELL:Right position: 16
      UMI:Right position: 26
      R2:Left position: 0
      Variants: 0
      Cost: 0
      CELL length: 16
      UMI length: 10
      R2 length: 55

>>>>>>>>>>>>>>>>> mixcr refineTagsAndSort <<<<<<<<<<<<<<<<<
Running:
mixcr refineTagsAndSort --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.refine.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.refine.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.refined.vdjca
Correction will be applied to the following tags: CELL, UMI
Sorting will be applied to the following tags: CELL, UMI
The following whitelist will be used for CELL: WhitelistFromAddress(address=builtin:737K-august-2016)
Initialization: progress unknown
Initialization: 5.4%
Initialization: 16.3%  ETA: 00:00:23
Initialization: 27.4%  ETA: 00:00:19
Initialization: 38.1%  ETA: 00:00:17
Initialization: 49.6%  ETA: 00:00:13
Initialization: 60%  ETA: 00:00:11
Initialization: 70.9%  ETA: 00:00:08
Initialization: 82%  ETA: 00:00:05
Initialization: 94.5%  ETA: 00:00:01
Counting CELL: 5.8%
Counting CELL: 16.8%  ETA: 00:00:07
Counting CELL: 35.5%  ETA: 00:00:03
Counting CELL: 58%  ETA: 00:00:01
Counting CELL: 76.6%  ETA: 00:00:01
Counting CELL: 100%  ETA: 00:00:00
Correcting CELL: 28.3%
Correcting CELL: 53.6%  ETA: 00:00:01
Correcting CELL: 73%  ETA: 00:00:01
Correcting CELL: 87.1%  ETA: 00:00:00
Correcting CELL: 97.2%  ETA: 00:00:06
Applying correction, sorting & filtering CELL: 9.4%
Applying correction, sorting & filtering CELL: 44.8%  ETA: 00:00:01
Applying correction, sorting & filtering CELL: 60.6%  ETA: 00:00:02
Applying correction, sorting & filtering CELL: 76.1%  ETA: 00:00:01
Writing CELL: 1.1%
Final sorting: 63.8%  ETA: 00:00:00
Writing correction result: ∞%  ETA: 00:00:00
Applying correction & sorting alignments by UMI: 0%
Applying correction & sorting alignments by UMI: 16.3%  ETA: 00:00:10
Applying correction & sorting alignments by UMI: 33%  ETA: 00:00:08
Applying correction & sorting alignments by UMI: 51.9%  ETA: 00:00:05
Applying correction & sorting alignments by UMI: 68.4%  ETA: 00:00:03
Applying correction & sorting alignments by UMI: 87.3%  ETA: 00:00:01
Sorting alignments by CELL: 3.1%
Writing result: 0%
Writing result: 96.3%  ETA: 00:00:00
================ report: refineTagsAndSort ================
Analysis time: 1.79m
Time spent in correction: 1.08m
Number of input records: 7559118
Number of output records: 86190 (1.14%)
CELL correction report:
  CELL input diversity: 220912
  CELL output diversity: 1817 (0.82%)
  CELL input reads: 7559118
  CELL output reads: 86190 (1.14%)
  CELL mean reads per tag: 34.22
  CELL input core diversity: 220219 (99.69%)
  CELL input core reads: 7558347 (99.99%)
  CELL directly corrected diversity: 42398 (19.19%)
  CELL directly corrected reads: 113649 (1.5%)
  CELL diversity filtered by tag quality: 42 (0.02%)
  CELL reads filtered by tag quality: 42 (0%)
  CELL diversity filtered by whitelist: 218537 (98.92%)
  CELL recursively corrected: 5614
UMI correction report:
  UMI input groups: 1817
  UMI input diversity: 40649
  UMI output diversity: 40451 (99.51%)
  UMI input reads: 86190
  UMI output reads: 86190 (100%)
  UMI mean reads per tag: 2.12
  UMI input core diversity: 40649 (100%)
  UMI input core reads: 86190 (100%)
  UMI directly corrected diversity: 198 (0.49%)
  UMI directly corrected reads: 199 (0.23%)
  UMI diversity filtered by tag quality: 0 (0%)
  UMI reads filtered by tag quality: 0 (0%)
  UMI diversity filtered by whitelist: 0 (0%)
  UMI recursively corrected: 0

>>>>>>>>>>>>>>>>>> mixcr assemblePartial <<<<<<<<<<<<<<<<<<
Running:
mixcr assemblePartial --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assemblePartial.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assemblePartial.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.refined.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679passembled.1.vdjca
Running assemble partial: 0%
Running assemble partial: 63.7%  ETA: 00:00:00
================= report: assemblePartial =================
Analysis time: 1.5s
Independent runs: 1817
Total alignments analysed: 86190
Number of output alignments: 86182 (99.99%)
Alignments already with CDR3 (no overlapping is performed): 10 (0.01%)
Successfully overlapped alignments: 8 (0.01%)
Left parts with too small N-region (failed to extract k-mer): 85 (0.1%)
Extracted k-mer diversity: 49900
Dropped due to wildcard in k-mer: 0 (0%)
Dropped due to too short NRegion parts in overlap: 3 (0%)
Dropped overlaps with empty N region due to no complete NDN coverage: 0 (0%)
Number of left-side alignments: 2150 (2.49%)
Number of right-side alignments: 256 (0.3%)
Complex overlaps: 0 (0%)
Over-overlaps: 0 (0%)
Partial alignments written to output: 86164 (99.97%)

>>>>>>>>>>>>>>>>>>>>>>> mixcr extend <<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr extend --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.extend.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.extend.report.json --threads 4 /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679passembled.1.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.extended.vdjca
Extending alignments: 0%
====================== report: extend ======================
Analysis time: 913ms
Extended alignments count: 3 (0%)
V extensions total: 3 (0%)
V extensions with merged targets: 0 (0%)
J extensions total: 0 (0%)
J extensions with merged targets: 0 (0%)
V+J extensions: 0 (0%)
Mean V extension length: 4.666666666666667
Mean J extension length: NaN

>>>>>>>>>>>>>>>>>>>>>> mixcr assemble <<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr assemble --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assemble.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assemble.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.extended.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.clna
Building pre-clones from tag groups: 0%
Building pre-clones from tag groups: 63.7%  ETA: 00:00:00
Assembling initial clonotypes: 0%
Initialization: progress unknown
Sorting alignments: 69.6%
Writing alignments: 69.1%
===================== report: assemble =====================
Analysis time: 3.23s
Final clonotype count: 7
Reads used in clonotypes, percent of total: 118 (0%)
Average number of reads per clonotype: 16.86
Reads dropped due to the lack of a clone sequence, percent of total: 86164 (0.02%)
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: 30 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Aligned reads processed: 148
Reads used in clonotypes before clustering, percent of total: 118 (0%)
Number of reads used as a core, percent of used: 118 (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%)
Clonotypes dropped as low quality: 0
Clonotypes eliminated by PCR error correction: 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: 4 (57.14%)
TRA non-functional: 1 (25%)
TRB chains: 2 (28.57%)
TRB non-functional: 0 (0%)
TRG chains: 1 (14.29%)
TRG non-functional: 1 (100%)
Pre-clone assembler report:
  Number of input groups: 1817
  Number of input groups with no assembling feature: 1806
  Number of input alignments: 86182
  Number of alignments with assembling feature: 18 (0.02%)
  Number of output pre-clones: 11
  Number of pre-clonotypes per group:   1
  Number of assembling feature sequences in groups with zero pre-clonotypes: 0
  Number of dropped pre-clones by tag suffix conflict: 0
  Number of dropped alignments by tag suffix conflict: 0
  Number of core alignments: 18 (0.02%)
  Discarded core alignments: 0 (0%)
  Empirically assigned alignments: 122 (0.14%)
  Empirical assignment conflicts: 0 (0%)
  Tag+VJ-gene empirically assigned alignments: 121 (0.14%)
  VJ-gene empirically assigned alignments: 1 (0%)
  Tag empirically assigned alignments: 0 (0%)
  Number of ambiguous groups: 0
  Number of ambiguous tag+V/J-gene combinations: 0
  Ignored non-productive alignments: 0 (0%)
  Unassigned alignments: 2554 (2.96%)

>>>>>>>>>>>>>>>>>> mixcr assembleContigs <<<<<<<<<<<<<<<<<<
Running:
mixcr assembleContigs --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assembleContigs.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.assembleContigs.report.json --threads 4 /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.clna /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.contigs.clns
Assembling contigs: 0%
================= report: assembleContigs =================
Analysis time: 631ms
Initial clonotype count: 7
Final clonotype count: 7 (100%)
Canceled assemblies: 0 (0%)
Number of premature termination assembly events, percent of number of initial clonotypes: 0.0 (0%)
Longest contig length: 424
Clustered variants: 0 (0%)
Reads in clustered variants: 0.0 (0%)
Reads in divided (newly created) clones: 0.0 (0%)
Clones with ambiguous letters in splitting region: 0 (0%)
Reads in clones with ambiguous letters in splitting region: 0.0 (0%)
Average number of ambiguous letters per clone with ambiguous letters in splitting region: NaN

>>>>>>>>>>>>>>>>>>>>>>>>> mixcr qc <<<<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr qc --print-to-stdout /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.contigs.clns /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.contigs.qc.txt

  Alignments dropped due to low sequence quality: 0.0% [OK]

>>>>>>>>>>>>>>>>>>>> mixcr exportClones <<<<<<<<<<<<<<<<<<<<
Running:
mixcr exportClones /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.contigs.clns /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848679/SRR11848679.clones.tsv
Exporting 
Clone splitting by Cell added automatically because -tags Cell field is present in the list.
Exporting clones: 0%
Analysis finished successfully.

>>>>>>>>>>>>>>>>>>>>>>> mixcr align <<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr align --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.align.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.align.report.json --threads 4 --preset 10x-sc-5gex --rna --species hsa /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE151177/SRR11848678/fastq/bamtofastq_R1.fastq.gz /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE151177/SRR11848678/fastq/bamtofastq_R2.fastq.gz /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.vdjca
The following tags and their roles will be associated with each output alignment:
  Payload tags: R2
  Cell tags: CELL(SQ)
  Molecule tags: UMI(SQ)
Alignment: 0%
Alignment: 1.2%  ETA: 02:43:22
Alignment: 2.4%  ETA: 02:46:08
Alignment: 3.7%  ETA: 02:26:51
Alignment: 5%  ETA: 02:22:02
Alignment: 6.3%  ETA: 02:28:35
Alignment: 7.7%  ETA: 02:15:36
Alignment: 8.9%  ETA: 02:29:42
Alignment: 10.2%  ETA: 02:19:13
Alignment: 11.4%  ETA: 02:23:39
Alignment: 12.7%  ETA: 02:12:17
Alignment: 14%  ETA: 02:17:28
Alignment: 15.2%  ETA: 02:14:35
Alignment: 16.5%  ETA: 02:08:03
Alignment: 17.8%  ETA: 02:06:33
Alignment: 19.1%  ETA: 02:02:48
Alignment: 20.6%  ETA: 01:53:19
Alignment: 21.9%  ETA: 01:54:24
Alignment: 23.4%  ETA: 01:45:08
Alignment: 24.8%  ETA: 01:42:28
Alignment: 26.2%  ETA: 01:51:44
Alignment: 27.3%  ETA: 02:03:49
Alignment: 28.7%  ETA: 01:48:14
Alignment: 30%  ETA: 01:43:33
Alignment: 31.3%  ETA: 01:48:59
Alignment: 32.6%  ETA: 01:43:27
Alignment: 33.8%  ETA: 01:46:10
Alignment: 35.1%  ETA: 01:41:12
Alignment: 36.3%  ETA: 01:45:04
Alignment: 37.7%  ETA: 01:33:06
Alignment: 38.9%  ETA: 01:38:08
Alignment: 40.2%  ETA: 01:34:00
Alignment: 41.4%  ETA: 01:34:06
Alignment: 42.7%  ETA: 01:29:15
Alignment: 44%  ETA: 01:27:31
Alignment: 45.4%  ETA: 01:17:05
Alignment: 46.7%  ETA: 01:19:45
Alignment: 48.2%  ETA: 01:11:04
Alignment: 49.7%  ETA: 01:08:16
Alignment: 50.9%  ETA: 01:17:23
Alignment: 52.1%  ETA: 01:21:02
Alignment: 53.4%  ETA: 01:10:55
Alignment: 54.8%  ETA: 01:06:09
Alignment: 56%  ETA: 01:10:54
Alignment: 57.4%  ETA: 01:03:15
Alignment: 58.6%  ETA: 01:08:51
Alignment: 59.9%  ETA: 01:01:47
Alignment: 61.1%  ETA: 01:02:16
Alignment: 62.4%  ETA: 00:58:14
Alignment: 63.7%  ETA: 00:58:21
Alignment: 64.9%  ETA: 00:56:39
Alignment: 66.2%  ETA: 00:50:58
Alignment: 67.5%  ETA: 00:52:23
Alignment: 68.8%  ETA: 00:48:15
Alignment: 70.1%  ETA: 00:44:45
Alignment: 71.5%  ETA: 00:39:40
Alignment: 72.9%  ETA: 00:38:34
Alignment: 74.4%  ETA: 00:34:52
Alignment: 75.8%  ETA: 00:36:05
Alignment: 77%  ETA: 00:38:28
Alignment: 78.2%  ETA: 00:35:03
Alignment: 79.6%  ETA: 00:28:25
Alignment: 80.9%  ETA: 00:30:07
Alignment: 82.2%  ETA: 00:27:47
Alignment: 83.4%  ETA: 00:26:15
Alignment: 84.7%  ETA: 00:23:31
Alignment: 86.1%  ETA: 00:21:07
Alignment: 87.3%  ETA: 00:20:36
Alignment: 88.6%  ETA: 00:17:57
Alignment: 89.8%  ETA: 00:16:20
Alignment: 91.1%  ETA: 00:13:23
Alignment: 92.4%  ETA: 00:12:26
Alignment: 93.7%  ETA: 00:09:35
Alignment: 95%  ETA: 00:07:51
Alignment: 96.5%  ETA: 00:04:39
Alignment: 97.8%  ETA: 00:03:12
Alignment: 99.3%  ETA: 00:00:59
====================== report: align ======================
Analysis time: 153.04m
Total sequencing reads: 525663031
Successfully aligned reads: 10445853 (1.99%)
Coverage (percent of successfully aligned):
  CDR3: 0%
  FR3_TO_FR4: 0%
  CDR2_TO_FR4: 0%
  FR2_TO_FR4: 0%
  CDR1_TO_FR4: 0%
  VDJRegion: 0%
Alignment failed: no hits (not TCR/IG?): 500476842 (95.21%)
Alignment failed: low total score: 14740336 (2.8%)
Overlapped: 0 (0%)
Overlapped and aligned: 0 (0%)
Overlapped and not aligned: 0 (0%)
Alignment-aided overlaps, percent of overlapped and aligned: 0 (NaN%)
No CDR3 parts alignments, percent of successfully aligned: 10361523 (99.19%)
Partial aligned reads, percent of successfully aligned: 84285 (0.81%)
Realigned with forced non-floating bound: 0 (0%)
Realigned with forced non-floating right bound in left read: 0 (0%)
Realigned with forced non-floating left bound in right read: 0 (0%)
TRA chains: 12093 (0.12%)
TRA non-functional: 16 (0.13%)
TRB chains: 820041 (7.85%)
TRB non-functional: 0 (0%)
TRD chains: 2340 (0.02%)
TRD non-functional: 0 (0%)
TRG chains: 6708723 (64.22%)
TRG non-functional: 1 (0%)
TRAD chains: 1013851 (9.71%)
TRAD non-functional: 0 (0%)
IGH chains: 644002 (6.17%)
IGH non-functional: 0 (0%)
IGK chains: 406283 (3.89%)
IGK non-functional: 0 (0%)
IGL chains: 838520 (8.03%)
IGL non-functional: 0 (0%)
Trimming report:
  R1 reads trimmed left: 281145 (0.05%)
  R1 reads trimmed right: 616165 (0.12%)
  Average R1 nucleotides trimmed left: 0.012055974314845817
  Average R1 nucleotides trimmed right: 0.031268257478049656
Tag parsing report:
  Execution time: 0ns
  Total reads: 525663031
  Matched reads: 525663031 (100%)
  Projection +R1 +R2: 525663031 (100%)
  For variant 0:
    For projection +R1 +R2:
      CELL:Left position: 0
      UMI:Left position: 16
      CELL:Right position: 16
      UMI:Right position: 26
      R2:Left position: 0
      Variants: 0
      Cost: 0
      CELL length: 16
      UMI length: 10
      R2 length: 57

>>>>>>>>>>>>>>>>> mixcr refineTagsAndSort <<<<<<<<<<<<<<<<<
Running:
mixcr refineTagsAndSort --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.refine.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.refine.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.refined.vdjca
Correction will be applied to the following tags: CELL, UMI
Sorting will be applied to the following tags: CELL, UMI
The following whitelist will be used for CELL: WhitelistFromAddress(address=builtin:737K-august-2016)
Initialization: progress unknown
Initialization: 3.4%
Initialization: 15.5%  ETA: 00:00:20
Initialization: 26.8%  ETA: 00:00:28
Initialization: 37.5%  ETA: 00:00:23
Initialization: 48.4%  ETA: 00:00:19
Initialization: 59.2%  ETA: 00:00:15
Initialization: 71%  ETA: 00:00:10
Initialization: 81.8%  ETA: 00:00:06
Initialization: 93.4%  ETA: 00:00:02
Counting CELL: 0.3%
Counting CELL: 18.5%  ETA: 00:00:14
Counting CELL: 34.4%  ETA: 00:00:04
Counting CELL: 49.1%  ETA: 00:00:03
Counting CELL: 73.6%  ETA: 00:00:02
Counting CELL: 89.8%  ETA: 00:00:00
Correcting CELL: 5.2%
Correcting CELL: 29.9%  ETA: 00:00:02
Correcting CELL: 50.2%  ETA: 00:00:02
Correcting CELL: 67.9%  ETA: 00:00:01
Correcting CELL: 82.7%  ETA: 00:00:01
Correcting CELL: 93.6%  ETA: 00:00:00
Applying #1 recursive correction for CELL: 0.6%
Applying correction, sorting & filtering CELL: 1.1%  ETA: 00:03:12
Applying correction, sorting & filtering CELL: 13.6%  ETA: 00:00:06
Applying correction, sorting & filtering CELL: 27.1%  ETA: 00:00:16
Applying correction, sorting & filtering CELL: 41.7%  ETA: 00:00:14
Applying correction, sorting & filtering CELL: 53.4%  ETA: 00:00:12
Applying correction, sorting & filtering CELL: 64.9%  ETA: 00:00:06
Applying correction, sorting & filtering CELL: 79.8%  ETA: 00:00:05
Applying correction, sorting & filtering CELL: 92.8%  ETA: 00:00:01
Writing CELL: 2.3%
Writing CELL: 18.8%  ETA: 00:00:09
Writing CELL: 30.5%  ETA: 00:00:17
Writing CELL: 43.2%  ETA: 00:00:09
Writing CELL: 54.4%  ETA: 00:00:08
Writing CELL: 67.8%  ETA: 00:00:07
Writing CELL: 80%  ETA: 00:00:03
Writing CELL: 95.4%  ETA: 00:00:00
Final sorting: 0%
Final sorting: 10.1%  ETA: 00:02:05
Final sorting: 20.1%  ETA: 00:01:51
Final sorting: 30.2%  ETA: 00:01:23
Final sorting: 40.7%  ETA: 00:01:08
Final sorting: 51.1%  ETA: 00:00:57
Final sorting: 61.5%  ETA: 00:00:48
Final sorting: 72%  ETA: 00:00:35
Final sorting: 82.7%  ETA: 00:00:19
Final sorting: 92.9%  ETA: 00:00:08
Writing correction result: ∞%  ETA: 00:00:00
Applying correction & sorting alignments by UMI: 0%
Applying correction & sorting alignments by UMI: 11.2%  ETA: 00:01:19
Applying correction & sorting alignments by UMI: 23.2%  ETA: 00:01:38
Applying correction & sorting alignments by UMI: 34.5%  ETA: 00:01:21
Applying correction & sorting alignments by UMI: 44.9%  ETA: 00:01:09
Applying correction & sorting alignments by UMI: 56.3%  ETA: 00:00:52
Applying correction & sorting alignments by UMI: 67.8%  ETA: 00:00:40
Applying correction & sorting alignments by UMI: 79.4%  ETA: 00:00:25
Applying correction & sorting alignments by UMI: 89.6%  ETA: 00:00:13
Applying correction & sorting alignments by UMI: 100%  ETA: 00:00:00
Sorting alignments by CELL: 0%
Sorting alignments by CELL: 12.3%  ETA: 00:01:25
Sorting alignments by CELL: 24.4%  ETA: 00:01:48
Sorting alignments by CELL: 37.3%  ETA: 00:01:24
Sorting alignments by CELL: 49.1%  ETA: 00:01:11
Sorting alignments by CELL: 59.3%  ETA: 00:00:56
Sorting alignments by CELL: 69.9%  ETA: 00:00:42
Sorting alignments by CELL: 80.4%  ETA: 00:00:25
Sorting alignments by CELL: 90.6%  ETA: 00:00:13
Writing result: 0%
Writing result: 10.1%  ETA: 00:01:56
Writing result: 20.2%  ETA: 00:01:42
Writing result: 30.5%  ETA: 00:01:22
Writing result: 40.6%  ETA: 00:01:11
Writing result: 51.4%  ETA: 00:01:03
Writing result: 62.3%  ETA: 00:00:45
Writing result: 72.6%  ETA: 00:00:35
Writing result: 82.9%  ETA: 00:00:20
Writing result: 93.1%  ETA: 00:00:08
================ report: refineTagsAndSort ================
Analysis time: 11.4m
Time spent in correction: 4.33m
Number of input records: 10445853
Number of output records: 10196745 (97.62%)
CELL correction report:
  CELL input diversity: 232663
  CELL output diversity: 110992 (47.71%)
  CELL input reads: 10445853
  CELL output reads: 10196757 (97.62%)
  CELL mean reads per tag: 44.9
  CELL input core diversity: 232214 (99.81%)
  CELL input core reads: 10445391 (100%)
  CELL directly corrected diversity: 60556 (26.03%)
  CELL directly corrected reads: 173801 (1.66%)
  CELL diversity filtered by tag quality: 23 (0.01%)
  CELL reads filtered by tag quality: 23 (0%)
  CELL diversity filtered by whitelist: 63468 (27.28%)
  CELL recursively corrected: 9031
UMI correction report:
  UMI input groups: 110992
  UMI input diversity: 4173065
  UMI output diversity: 4135809 (99.11%)
  UMI input reads: 10196757
  UMI output reads: 10196745 (100%)
  UMI mean reads per tag: 2.44
  UMI input core diversity: 4173037 (100%)
  UMI input core reads: 10196729 (100%)
  UMI directly corrected diversity: 37244 (0.89%)
  UMI directly corrected reads: 37840 (0.37%)
  UMI diversity filtered by tag quality: 12 (0%)
  UMI reads filtered by tag quality: 12 (0%)
  UMI diversity filtered by whitelist: 0 (0%)
  UMI recursively corrected: 56

>>>>>>>>>>>>>>>>>> mixcr assemblePartial <<<<<<<<<<<<<<<<<<
Running:
mixcr assemblePartial --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assemblePartial.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assemblePartial.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.refined.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678passembled.1.vdjca
Running assemble partial: 0%
Running assemble partial: 10.5%  ETA: 00:01:42
Running assemble partial: 20.7%  ETA: 00:01:17
Running assemble partial: 31.5%  ETA: 00:01:09
Running assemble partial: 42.2%  ETA: 00:00:59
Running assemble partial: 52.7%  ETA: 00:00:45
Running assemble partial: 63%  ETA: 00:00:39
Running assemble partial: 73.4%  ETA: 00:00:28
Running assemble partial: 84.3%  ETA: 00:00:17
Running assemble partial: 94.4%  ETA: 00:00:06
================= report: assemblePartial =================
Analysis time: 1.75m
Independent runs: 110992
Total alignments analysed: 10196745
Number of output alignments: 10196723 (100%)
Alignments already with CDR3 (no overlapping is performed): 82 (0%)
Successfully overlapped alignments: 22 (0%)
Left parts with too small N-region (failed to extract k-mer): 803 (0.01%)
Extracted k-mer diversity: 4898757
Dropped due to wildcard in k-mer: 0 (0%)
Dropped due to too short NRegion parts in overlap: 11 (0%)
Dropped overlaps with empty N region due to no complete NDN coverage: 0 (0%)
Number of left-side alignments: 214664 (2.11%)
Number of right-side alignments: 13305 (0.13%)
Complex overlaps: 0 (0%)
Over-overlaps: 0 (0%)
Partial alignments written to output: 10196619 (100%)

>>>>>>>>>>>>>>>>>>>>>>> mixcr extend <<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr extend --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.extend.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.extend.report.json --threads 4 /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678passembled.1.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.extended.vdjca
Extending alignments: 0%
Extending alignments: 11.9%  ETA: 00:00:14
Extending alignments: 26.1%  ETA: 00:00:10
Extending alignments: 38.9%  ETA: 00:00:23
Extending alignments: 51.7%  ETA: 00:00:25
Extending alignments: 63%  ETA: 00:00:09
Extending alignments: 75.9%  ETA: 00:00:05
Extending alignments: 89.8%  ETA: 00:00:02
====================== report: extend ======================
Analysis time: 2.22m
Extended alignments count: 49 (0%)
V extensions total: 4 (0%)
V extensions with merged targets: 0 (0%)
J extensions total: 45 (0%)
J extensions with merged targets: 0 (0%)
V+J extensions: 0 (0%)
Mean V extension length: 2.5
Mean J extension length: 9.8

>>>>>>>>>>>>>>>>>>>>>> mixcr assemble <<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr assemble --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assemble.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assemble.report.json /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.extended.vdjca /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.clna
Building pre-clones from tag groups: 0%
Building pre-clones from tag groups: 10.5%  ETA: 00:01:59
Building pre-clones from tag groups: 21.1%  ETA: 00:01:29
Building pre-clones from tag groups: 31.4%  ETA: 00:01:13
Building pre-clones from tag groups: 41.5%  ETA: 00:01:03
Building pre-clones from tag groups: 51.8%  ETA: 00:00:51
Building pre-clones from tag groups: 62.5%  ETA: 00:00:38
Building pre-clones from tag groups: 73.4%  ETA: 00:00:26
Building pre-clones from tag groups: 84%  ETA: 00:00:16
Building pre-clones from tag groups: 94.2%  ETA: 00:00:06
Initialization: progress unknown
Initialization: progress unknown
Sorting alignments: 1.1%
Sorting alignments: 12.2%  ETA: 00:01:10
Sorting alignments: 23.3%  ETA: 00:01:02
Sorting alignments: 34%  ETA: 00:00:55
Sorting alignments: 44.9%  ETA: 00:00:45
Sorting alignments: 55%  ETA: 00:00:35
Sorting alignments: 65.9%  ETA: 00:00:28
Sorting alignments: 76.3%  ETA: 00:00:20
Sorting alignments: 87.2%  ETA: 00:00:10
Sorting alignments: 97.6%  ETA: 00:00:01
Writing alignments: 0.3%
Writing alignments: 10.5%  ETA: 00:01:19
Writing alignments: 21%  ETA: 00:01:07
Writing alignments: 31.8%  ETA: 00:00:56
Writing alignments: 42.1%  ETA: 00:00:50
Writing alignments: 52.5%  ETA: 00:00:41
Writing alignments: 62.9%  ETA: 00:00:32
Writing alignments: 73.3%  ETA: 00:00:23
Writing alignments: 83.8%  ETA: 00:00:13
Writing alignments: 94.1%  ETA: 00:00:05
===================== report: assemble =====================
Analysis time: 3.21m
Final clonotype count: 18
Reads used in clonotypes, percent of total: 787 (0%)
Average number of reads per clonotype: 43.72
Reads dropped due to the lack of a clone sequence, percent of total: 10196620 (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: 25 (0%)
Reads dropped due to failed mapping, percent of total: 44 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Aligned reads processed: 856
Reads used in clonotypes before clustering, percent of total: 787 (0%)
Number of reads used as a core, percent of used: 787 (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%)
Clonotypes dropped as low quality: 0
Clonotypes eliminated by PCR error correction: 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: 14 (77.78%)
TRA non-functional: 7 (50%)
TRB chains: 1 (5.56%)
TRB non-functional: 0 (0%)
TRG chains: 3 (16.67%)
TRG non-functional: 1 (33.33%)
Pre-clone assembler report:
  Number of input groups: 110992
  Number of input groups with no assembling feature: 110964
  Number of input alignments: 10196723
  Number of alignments with assembling feature: 103 (0%)
  Number of output pre-clones: 28
  Number of pre-clonotypes per group:   1
  Number of assembling feature sequences in groups with zero pre-clonotypes: 0
  Number of dropped pre-clones by tag suffix conflict: 0
  Number of dropped alignments by tag suffix conflict: 0
  Number of core alignments: 103 (0%)
  Discarded core alignments: 0 (0%)
  Empirically assigned alignments: 731 (0.01%)
  Empirical assignment conflicts: 0 (0%)
  Tag+VJ-gene empirically assigned alignments: 433 (0%)
  VJ-gene empirically assigned alignments: 298 (0%)
  Tag empirically assigned alignments: 0 (0%)
  Number of ambiguous groups: 0
  Number of ambiguous tag+V/J-gene combinations: 0
  Ignored non-productive alignments: 0 (0%)
  Unassigned alignments: 188808 (1.85%)

>>>>>>>>>>>>>>>>>> mixcr assembleContigs <<<<<<<<<<<<<<<<<<
Running:
mixcr assembleContigs --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assembleContigs.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.assembleContigs.report.json --threads 4 /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.clna /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.contigs.clns
Assembling contigs: 0%
================= report: assembleContigs =================
Analysis time: 853ms
Initial clonotype count: 18
Final clonotype count: 18 (100%)
Canceled assemblies: 0 (0%)
Number of premature termination assembly events, percent of number of initial clonotypes: 0.0 (0%)
Longest contig length: 393
Clustered variants: 0 (0%)
Reads in clustered variants: 0.0 (0%)
Reads in divided (newly created) clones: 0.0 (0%)
Clones with ambiguous letters in splitting region: 0 (0%)
Reads in clones with ambiguous letters in splitting region: 0.0 (0%)
Average number of ambiguous letters per clone with ambiguous letters in splitting region: NaN

>>>>>>>>>>>>>>>>>>>>>>>>> mixcr qc <<<<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr qc --print-to-stdout /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.contigs.clns /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.contigs.qc.txt

  Alignments dropped due to low sequence quality: 4.75% [OK]

>>>>>>>>>>>>>>>>>>>> mixcr exportClones <<<<<<<<<<<<<<<<<<<<
Running:
mixcr exportClones /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.contigs.clns /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE151177/SRR11848678/SRR11848678.clones.tsv
Exporting 
Clone splitting by Cell added automatically because -tags Cell field is present in the list.
Analysis finished successfully.

[mizraelson]

A low number of successfully aligned reads is expected for non-enriched 3' 10x data.

[malonzm1]

Thanks. What about GSE160097 which used PBMCs and 5' 10x VDJ?

>>>>>>>>>>>>>>>>>>>>>>> mixcr align <<<<<<<<<<<<<<<<<<<<<<<
Running:
mixcr align --report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE160097/SRR12898573.2/SRR12898573.align.report.txt --json-report /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE160097/SRR12898573.2/SRR12898573.align.report.json --threads 4 --preset 10x-sc-xcr-vdj --save-output-file-names /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE160097/SRR12898573.2/SRR12898573.align.list --rna --species hsa /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE160097/SRR12898573/fastq/bamtofastq_R1.fastq.gz /scratch/cs/pan-autoimmune/data/raw_data/scRNAseq/GSE160097/SRR12898573/fastq/bamtofastq_R2.fastq.gz /scratch/cs/pan-autoimmune/data/mixcr/scRNAseq/GSE160097/SRR12898573.2/SRR12898573.vdjca
The following tags and their roles will be associated with each output alignment:
  Payload tags: R2
  Cell tags: CELL(SQ)
  Molecule tags: UMI(SQ)
Alignment: 0%
Alignment: 2.4%  ETA: 01:21:09
Alignment: 4.8%  ETA: 01:17:53
Alignment: 7.4%  ETA: 01:12:51
Alignment: 9.9%  ETA: 01:10:51
Alignment: 12.5%  ETA: 01:07:48
Alignment: 15.1%  ETA: 01:06:48
Alignment: 17.6%  ETA: 01:05:51
Alignment: 20%  ETA: 01:07:02
Alignment: 22.8%  ETA: 00:53:59
Alignment: 25.6%  ETA: 00:53:28
Alignment: 28%  ETA: 00:59:35
Alignment: 30.4%  ETA: 00:57:27
Alignment: 32.9%  ETA: 00:54:41
Alignment: 35.4%  ETA: 00:52:05
Alignment: 37.9%  ETA: 00:48:50
Alignment: 40.4%  ETA: 00:47:03
Alignment: 42.8%  ETA: 00:48:20
Alignment: 45.3%  ETA: 00:43:53
Alignment: 48.1%  ETA: 00:36:33
Alignment: 50.8%  ETA: 00:36:37
Alignment: 53.3%  ETA: 00:38:14
Alignment: 55.7%  ETA: 00:35:44
Alignment: 58.3%  ETA: 00:33:11
Alignment: 60.9%  ETA: 00:30:00
Alignment: 63.4%  ETA: 00:28:34
Alignment: 66.1%  ETA: 00:25:41
Alignment: 68.5%  ETA: 00:25:50
Alignment: 71%  ETA: 00:22:53
Alignment: 74.1%  ETA: 00:17:08
Alignment: 76.5%  ETA: 00:19:19
Alignment: 78.9%  ETA: 00:17:38
Alignment: 81.4%  ETA: 00:14:42
Alignment: 84%  ETA: 00:12:37
Alignment: 86.5%  ETA: 00:10:37
Alignment: 89.1%  ETA: 00:08:26
Alignment: 91.6%  ETA: 00:06:38
Alignment: 93.9%  ETA: 00:05:22
Alignment: 96.6%  ETA: 00:02:34
Alignment: 99.5%  ETA: 00:00:18
====================== report: align ======================
Analysis time: 78.39m
Total sequencing reads: 249190071
Successfully aligned reads: 1906850 (0.77%)
Coverage (percent of successfully aligned):
  CDR3: 0.03%
  FR3_TO_FR4: 0%
  CDR2_TO_FR4: 0%
  FR2_TO_FR4: 0%
  CDR1_TO_FR4: 0%
  VDJRegion: 0%
Alignment failed: no hits (not TCR/IG?): 243301880 (97.64%)
Alignment failed: low total score: 3981341 (1.6%)
Overlapped: 0 (0%)
Overlapped and aligned: 0 (0%)
Overlapped and not aligned: 0 (0%)
Alignment-aided overlaps, percent of overlapped and aligned: 0 (NaN%)
No CDR3 parts alignments, percent of successfully aligned: 1831620 (96.05%)
Partial aligned reads, percent of successfully aligned: 74598 (3.91%)
Realigned with forced non-floating bound: 0 (0%)
Realigned with forced non-floating right bound in left read: 0 (0%)
Realigned with forced non-floating left bound in right read: 0 (0%)
TRA chains: 16607 (0.87%)
TRA non-functional: 158 (0.95%)
TRB chains: 543345 (28.49%)
TRB non-functional: 1 (0%)
TRD chains: 220 (0.01%)
TRD non-functional: 0 (0%)
TRG chains: 205928 (10.8%)
TRG non-functional: 4 (0%)
IGH chains: 209685 (11%)
IGH non-functional: 0 (0%)
TRAD chains: 614588 (32.23%)
TRAD non-functional: 0 (0%)
IGK chains: 113804 (5.97%)
IGK non-functional: 0 (0%)
IGL chains: 202673 (10.63%)
IGL non-functional: 0 (0%)
Trimming report:
  R1 reads trimmed left: 41986 (0.02%)
  R1 reads trimmed right: 9703 (0%)
  Average R1 nucleotides trimmed left: 0.002577277647631474
  Average R1 nucleotides trimmed right: 0.001094558057251005
Tag parsing report:
  Execution time: 0ns
  Total reads: 249190071
  Matched reads: 249190071 (100%)
  Projection +R1 +R2: 249190071 (100%)
  For variant 0:
    For projection +R1 +R2:
      CELL:Left position: 0
      UMI:Left position: 16
      CELL:Right position: 16
      UMI:Right position: 26
      R2:Left position: 0
      Variants: 0
      Cost: 0
      CELL length: 16
      UMI length: 10
      R2 length:
        64~97: + 16992333 (6.82%) = 16992333 (6.82%)
        98: + 230774605 (92.61%) = 247766938 (99.43%)
        99~115: + 1423133 (0.57%) = 249190071 (100%)

[mizraelson]

This file seems to be from gene expression not VDJ targeted library. The authors did both in the paper.
https://trace.ncbi.nlm.nih.gov/Traces/?view=run_browser&acc=SRR12898573&display=metadata

For 10x VDJ the read length should be 150+150 according to the publication.

[malonzm1]

Thanks. I actually emailed the NCBI GEO contact who told me they were all from VDJ sequencing (I guess this was not accurate). Is this the paper you are referring to https://onlinelibrary.wiley.com/doi/10.1002/eji.202048797?

[mizraelson]

Here in the description to the file it is said:
"The sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5' GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for TCR libraries (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in). "

Then I checked the read length for that file here and it is 98, which means its a 5' GEX.