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milaboratory / mixcr

MiXCR is an ultimate software platform for analysis of Next-Generation Sequencing (NGS) data for immune profiling.
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I am new in using mixcr but I encounter a problem I can not solve #1252

Closed A-legac45 closed 1 year ago

A-legac45 commented 1 year ago

Hello,

I am new in using mixcr but I encounter a problem I can not solve

I have bulk tcrseq data paried end and I am using the following command which do not work

thanks for your help

/bioinfo/local/build/Centos/MiXCR/mixcr-3.0.12/mixcr analyze amplicon --species hsa \ --starting-material rna \ --receptor-type TRA \ --5-end v-primers \ --3-end j-primers \ --export "-p full" \ --verbose \ --adapters adapters-present \ /data/kdi_prod/dataset_all/2017669/export/user/D1454T01/D1454T01.R1.fastq.gz /data/kdi_prod/dataset_all/2017669/export/user/D1454T01/D1454T01.R2.fastq.gz /data/users/alegac/kdi_2017669/output_prefix

Alignment: 0% /bioinfo/local/build/Centos/MiXCR/mixcr-3.0.12/mixcr: line 196: 41913 Killed $java -D${app}.path="$dir" -D${app}.command=scr "${javaArgs[@]}" -jar "$jar" "${appArgs[@]}"

mizraelson commented 1 year ago

Hi, can you please reproduce the error using the latest MiXCR version? I can help you with the command if needed.

A-legac45 commented 1 year ago

Hi many thanks,

I activate a newer version of mixcr but now it is worse I have to adapt the parameters I imagine

License activated successfully. (base) alegac@ICR-R032MGH0XP ~ % /Users/alegac/opt/anaconda3/pkgs/mixcr-4.4.1-0/bin/mixcr analyze amplicon --species hsa \ --starting-material rna \ --receptor-type TRA \ --5-end v-primers \ --3-end j-primers \ --export "-p full" \ --verbose \ --adapters adapters-present \/Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R1.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R2.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/results

4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 Unknown options: '--starting-material', '--receptor-type', '--5-end', 'v-primers', '--3-end', 'j-primers', '--export', '-p full', '--adapters', 'adapters-present', '/Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R1.fastq.gz', '/Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R2.fastq.gz', '/Users/alegac/Documents/bulk_tcr/D1454T02/results

A-legac45 commented 1 year ago

I am trying this is which seems to start running I let you known if it work

/Users/alegac/opt/anaconda3/pkgs/mixcr-4.4.1-0/bin/mixcr analyze milab-human-rna-tcr-umi-multiplex /Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R1.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R2.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/results

Is it correct to use this pre analyse pipeline for a data obtained taht way, expected size 150pb used kit for index Dual Indexes or IDT for Illumina - DNA/RNA #20027213.

Many thanks for your help

mizraelson commented 1 year ago

Did you use the TCR multiplex kit from Milaboratory?

A-legac45 commented 1 year ago

I am not sure I did not realize the experiment

the use is the following

Tube I7_Index_sequence I5_Index_sequence 1 TCCATTGCCG GAATGCACGA 2 ACGCCTTGTT TAAGGAACGT 3 GATCAAGGCA CCTTGTTAAT 4 CGGTTACGGC AAGACTATAG 5 TTCTACATAC CTAACTGTAA 6 ACAGTGTATG GAACATACGG

The protocol The biologist gave me is from milaboratory so I expect yes HUMAN TCR RNA MULTIPLEX TCR α and β repertoires with UMI User Manual v.1.11

A-legac45 commented 1 year ago

This is the output file I got : results.align.report.txt

I got this message

Not enough memory for run command, try to increase -Xmx. Available memory: 32768 Mb Example: mixcr -Xmx40g analyze milab-human-rna-tcr-umi-multiplex /Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R1.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/D1454T02_trimmed_R2.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T02/results This run used approximately 1438m of memory

mizraelson commented 1 year ago

It means that Mixcr requires more memory to process the data. Depending on the dataset it might need more or less memory. How large are your files?

mizraelson commented 1 year ago

Also, one more question, is your data already demultiplexed by samples?

A-legac45 commented 1 year ago

My files are 3.3 and 2.9 go for R1 and R2 how much should I try ? Do you know?

mizraelson commented 1 year ago

Try -Xmx15G. That should be enough.

A-legac45 commented 1 year ago

thanks

mizraelson commented 1 year ago

Let me know how it works

A-legac45 commented 1 year ago

Hello,

Thanks a lot for your answer, help and quick responses. I am wondering if this kind of QC report if usual?

I got warning and alert for those criteria for the two samples I try until nown :

Barcode collisions in clonotype assembly: 2.34% [WARN] Reads used in clonotypes: 89.15% [WARN] UMIs artificial diversity eliminated: 80.34% [ALERT]

Best regards,

Anne-Laure LE GAC

Post-doctorant

Equipe Lantz - INSERM U932, Immunité et Cancer Centre immunothérapie, 2ème étage - burreau 2a-20 26 rue d'Ulm, 75005 PARIS

Téléphone : +33 (0)1 44 32 68 34

De : mizraelson @.***> Envoyé : vendredi 21 juillet 2023 20:07:25 À : milaboratory/mixcr Cc : Le Gac Anne-Laure; Author Objet : Re: [milaboratory/mixcr] I am new in using mixcr but I encounter a problem I can not solve (Issue #1252)

Let me know how it works

— Reply to this email directly, view it on GitHubhttps://github.com/milaboratory/mixcr/issues/1252#issuecomment-1646067724, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AYDTLNRLFVDWCKHDOOHQBNLXRLAN3ANCNFSM6AAAAAA2S2KHNY. You are receiving this because you authored the thread.Message ID: @.***>

Successfully aligned reads: 95.47% [OK] Off target (non TCR/IG) reads: 0.59% [OK] Reads with no V or J hits: 1.99% [OK] Reads with no barcode: 1.49% [OK] Overlapped paired-end reads: 94.90% [OK] Alignments that do not cover CDR3: 0.51% [OK] Tag groups that do not cover CDR3: 0.067% [OK] Barcode collisions in clonotype assembly: 2.013% [WARN] Unassigned alignments in clonotype assembly: 3.27% [OK] Reads used in clonotypes: 89.86% [WARN] Alignments dropped due to low sequence quality: 0.0% [OK] Alignments clustered in PCR error correction: 0.16% [OK] Clonotypes clustered in PCR error correction: 0.52% [OK] Clones dropped in post-filtering: 0.0% [OK] Alignments dropped in clones post-filtering: 0.0% [OK] Reads dropped in tags error correction and filtering: 2.44% [OK] UMIs artificial diversity eliminated: 75.97% [ALERT] Reads dropped in UMI error correction and whitelist: 0.14% [OK] Reads dropped in tags filtering: 2.29% [OK]

Successfully aligned reads: 94.94% [OK] Off target (non TCR/IG) reads: 0.61% [OK] Reads with no V or J hits: 2.071% [OK] Reads with no barcode: 1.74% [OK] Overlapped paired-end reads: 94.50% [OK] Alignments that do not cover CDR3: 1.26% [OK] Tag groups that do not cover CDR3: 0.11% [OK] Barcode collisions in clonotype assembly: 2.34% [WARN] Unassigned alignments in clonotype assembly: 4.030% [OK] Reads used in clonotypes: 89.15% [WARN] Alignments dropped due to low sequence quality: 1.50% [OK] Alignments clustered in PCR error correction: 0.092% [OK] Clonotypes clustered in PCR error correction: 0.29% [OK] Clones dropped in post-filtering: 0.0% [OK] Alignments dropped in clones post-filtering: 0.0% [OK] Reads dropped in tags error correction and filtering: 1.98% [OK] UMIs artificial diversity eliminated: 80.34% [ALERT] Reads dropped in UMI error correction and whitelist: 0.10% [OK] Reads dropped in tags filtering: 1.87% [OK]

mizraelson commented 1 year ago

Hi,

If you're able to share the report files generated by mixcr, I may be able to provide more detailed insights.

Sincerely, Mark

A-legac45 commented 1 year ago

thanks for your feedback ,

it is the first time I am running mixcr and I want to be sure I making things properly. In my previous message I join the qc_report in txt.

I am copy pasting it here maybe it is easier to look at.

sample 1

Successfully aligned reads: 95.47% [OK] Off target (non TCR/IG) reads: 0.59% [OK] Reads with no V or J hits: 1.99% [OK] Reads with no barcode: 1.49% [OK] Overlapped paired-end reads: 94.90% [OK] Alignments that do not cover CDR3: 0.51% [OK] Tag groups that do not cover CDR3: 0.067% [OK] Barcode collisions in clonotype assembly: 2.013% [WARN] Unassigned alignments in clonotype assembly: 3.27% [OK] Reads used in clonotypes: 89.86% [WARN] Alignments dropped due to low sequence quality: 0.0% [OK] Alignments clustered in PCR error correction: 0.16% [OK] Clonotypes clustered in PCR error correction: 0.52% [OK] Clones dropped in post-filtering: 0.0% [OK] Alignments dropped in clones post-filtering: 0.0% [OK] Reads dropped in tags error correction and filtering: 2.44% [OK] UMIs artificial diversity eliminated: 75.97% [ALERT] Reads dropped in UMI error correction and whitelist: 0.14% [OK] Reads dropped in tags filtering: 2.29% [OK]

SAMPLE 2

Successfully aligned reads: 94.94% [OK] Off target (non TCR/IG) reads: 0.61% [OK] Reads with no V or J hits: 2.071% [OK] Reads with no barcode: 1.74% [OK] Overlapped paired-end reads: 94.50% [OK] Alignments that do not cover CDR3: 1.26% [OK] Tag groups that do not cover CDR3: 0.11% [OK] Barcode collisions in clonotype assembly: 2.34% [WARN] Unassigned alignments in clonotype assembly: 4.030% [OK] Reads used in clonotypes: 89.15% [WARN] Alignments dropped due to low sequence quality: 1.50% [OK] Alignments clustered in PCR error correction: 0.092% [OK] Clonotypes clustered in PCR error correction: 0.29% [OK] Clones dropped in post-filtering: 0.0% [OK] Alignments dropped in clones post-filtering: 0.0% [OK] Reads dropped in tags error correction and filtering: 1.98% [OK] UMIs artificial diversity eliminated: 80.34% [ALERT] Reads dropped in UMI error correction and whitelist: 0.10% [OK] Reads dropped in tags filtering: 1.87% [OK]

Many thanks

best regards,

Anne-Laure LE GAC

Post-doctorant

Equipe Lantz - INSERM U932, Immunité et Cancer Centre immunothérapie, 2ème étage - burreau 2a-20 26 rue d'Ulm, 75005 PARIS

Téléphone : +33 (0)1 44 32 68 34

De : mizraelson @.***> Envoyé : mercredi 26 juillet 2023 17:55:10 À : milaboratory/mixcr Cc : Le Gac Anne-Laure; Author Objet : Re: [milaboratory/mixcr] I am new in using mixcr but I encounter a problem I can not solve (Issue #1252)

Hi,

If you're able to share the report files generated by mixcr, I may be able to provide more detailed insights.

Sincerely, Mark

— Reply to this email directly, view it on GitHubhttps://github.com/milaboratory/mixcr/issues/1252#issuecomment-1652094749, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AYDTLNXB3SSFGHNYSZMBEITXSE4V5ANCNFSM6AAAAAA2S2KHNY. You are receiving this because you authored the thread.Message ID: @.***>

mizraelson commented 1 year ago

Hi, sorry I meant align, refine and assemble txt reports. But from what I see here everything seems fine.

A-legac45 commented 1 year ago

Hi,

Many thanks for your answers,

I nearly got all my results nown, I just have a problem with two of my samples for which I got this message :

writing pre-clones: progress unknown Writing pre-clones: 0,4% Writing pre-clones: 10,6% ETA: 00:04:46 Writing pre-clones: 20,9% ETA: 00:04:08 Writing pre-clones: 31,2% ETA: 00:03:49 Writing pre-clones: 41,4% ETA: 00:03:58 Writing pre-clones: 51,9% ETA: 00:02:36 Writing pre-clones: 61,9% ETA: 00:01:57 Writing pre-clones: 72% ETA: 00:01:30 Writing pre-clones: 82,2% ETA: 00:01:00 Writing pre-clones: 92,7% ETA: 00:00:22 Writing pre-clones: 77,1% Initialization: progress unknown Mapping low quality reads: 5,6% 4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 picocli.CommandLine$ExecutionException: Error while running command assemble java.lang.RuntimeException: Missing event detected. at com.milaboratory.mixcr.cli.Main.registerExceptionHandlers$lambda-12(SourceFile:323) at picocli.CommandLine.execute(CommandLine.java:2088) at com.milaboratory.mixcr.cli.CommandAnalyze$Cmd$PlanBuilder.executeSteps(SourceFile:470) at com.milaboratory.mixcr.cli.CommandAnalyze$Cmd.run0(SourceFile:412) at com.milaboratory.mixcr.cli.MiXCRCommand.run(SourceFile:37) at picocli.CommandLine.executeUserObject(CommandLine.java:1939) at picocli.CommandLine.access$1300(CommandLine.java:145) at picocli.CommandLine$RunLast.executeUserObjectOfLastSubcommandWithSameParent(CommandLine.java:2358) at picocli.CommandLine$RunLast.handle(CommandLine.java:2352) at picocli.CommandLine$RunLast.handle(CommandLine.java:2314) at picocli.CommandLine$AbstractParseResultHandler.execute(CommandLine.java:2179) at picocli.CommandLine$RunLast.execute(CommandLine.java:2316) at com.milaboratory.mixcr.cli.Main.registerLogger$lambda-26(SourceFile:426) at picocli.CommandLine.execute(CommandLine.java:2078) at com.milaboratory.mixcr.cli.Main.main(SourceFile:98) Caused by: java.lang.RuntimeException: Missing event detected. at com.milaboratory.o.hN.a(SourceFile:59) at com.milaboratory.o.hT$c.accept(SourceFile:1484) at cc.redberry.pipe.blocks.FilteringPort.take(FilteringPort.java:65) at cc.redberry.pipe.blocks.O2ITransmitter.run(O2ITransmitter.java:63) at java.base/java.lang.Thread.run(Thread.java:829) Mapping low quality reads: 47,2% ETA: 00:00:01

the qualitty control of this sample is

[cid:2db7b801-7c20-469f-96dc-3e8956067129]

and the two results files I add from mixcr are the following (in attach)

How can I solve this problem?

Thanks a lot for your help

Anne-Laure LE GAC

Post-doctorant

Equipe Lantz - INSERM U932, Immunité et Cancer Centre immunothérapie, 2ème étage - burreau 2a-20 26 rue d'Ulm, 75005 PARIS

Téléphone : +33 (0)1 44 32 68 34

De : mizraelson @.***> Envoyé : mercredi 26 juillet 2023 20:06:39 À : milaboratory/mixcr Cc : Le Gac Anne-Laure; Author Objet : Re: [milaboratory/mixcr] I am new in using mixcr but I encounter a problem I can not solve (Issue #1252)

Hi, sorry I meant align, refine and assemble txt reports. But from what I see here everything seems fine.

— Reply to this email directly, view it on GitHubhttps://github.com/milaboratory/mixcr/issues/1252#issuecomment-1652271822, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AYDTLNVAXMRRINEVIUZETUTXSFMC7ANCNFSM6AAAAAA2S2KHNY. You are receiving this because you authored the thread.Message ID: @.***>

Analysis date: Sat Jul 29 10:57:44 CEST 2023 Input file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Output file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refined.vdjca Version: 4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 Command line arguments: refineTagsAndSort --report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refine.report.txt --json-report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refine.report.json /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refined.vdjca Analysis time: 34,78m Time spent in correction: 22,65m Number of input records: 42577778 Number of output records: 41901623 (98,41%) UMI correction report: UMI input diversity: 2357409 UMI output diversity: 396970 (16,84%) UMI input reads: 42577778 UMI output reads: 42516213 (99,86%) UMI mean reads per tag: 18,06 UMI input core diversity: 1144102 (48,53%) UMI input core reads: 41363757 (97,15%) UMI directly corrected diversity: 1898875 (80,55%) UMI directly corrected reads: 3133527 (7,36%) UMI diversity filtered by tag quality: 61564 (2,61%) UMI reads filtered by tag quality: 61565 (0,14%) UMI diversity filtered by whitelist: 0 (0%) UMI recursively corrected: 686575 Filter report: Number of groups: 396970 Number of groups accepted: 233083 (58,72%) Total records weight: 42516213 Records weight accepted: 41901623 (98,55%) Operator #0: Effective threshold: 10.0 Nested thresholds:

0: 10

  #1: 127

====================================== Analysis date: Mon Jul 31 09:19:33 CEST 2023 Input file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Output file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refined.vdjca Version: 4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 Command line arguments: refineTagsAndSort --report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refine.report.txt --json-report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refine.report.json /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.refined.vdjca Analysis time: 37,19m Time spent in correction: 23,51m Number of input records: 42577778 Number of output records: 41901623 (98,41%) UMI correction report: UMI input diversity: 2357409 UMI output diversity: 396970 (16,84%) UMI input reads: 42577778 UMI output reads: 42516213 (99,86%) UMI mean reads per tag: 18,06 UMI input core diversity: 1144102 (48,53%) UMI input core reads: 41363757 (97,15%) UMI directly corrected diversity: 1898875 (80,55%) UMI directly corrected reads: 3133527 (7,36%) UMI diversity filtered by tag quality: 61564 (2,61%) UMI reads filtered by tag quality: 61565 (0,14%) UMI diversity filtered by whitelist: 0 (0%) UMI recursively corrected: 686575 Filter report: Number of groups: 396970 Number of groups accepted: 233083 (58,72%) Total records weight: 42516213 Records weight accepted: 41901623 (98,55%) Operator #0: Effective threshold: 10.0 Nested thresholds:

0: 10

  #1: 127

======================================

Analysis date: Sat Jul 29 09:37:50 CEST 2023 Input file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R1.fastq.gz,/Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R2.fastq.gz Output file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Version: 4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 Command line arguments: align --report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.align.report.txt --json-report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.align.report.json --preset milab-human-rna-tcr-umi-multiplex /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R1.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R2.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Analysis time: 45,1m Total sequencing reads: 45247538 Successfully aligned reads: 42581901 (94,11%) Coverage (percent of successfully aligned): CDR3: 99,87% 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?): 378238 (0,84%) Alignment failed: absence of V hits: 695317 (1,54%) Alignment failed: absence of J hits: 509594 (1,13%) Alignment failed: no target with both V and J alignments: 142309 (0,31%) Alignment failed: absent barcode: 940179 (2,08%) Overlapped: 42961629 (94,95%) Overlapped and aligned: 42216775 (93,3%) Overlapped and not aligned: 744854 (1,65%) Alignment-aided overlaps, percent of overlapped and aligned: 79675 (0,19%) No CDR3 parts alignments, percent of successfully aligned: 182 (0%) Partial aligned reads, percent of successfully aligned: 56224 (0,13%) V gene chimeras: 223 (0%) J gene chimeras: 6777 (0,01%) Paired-end alignment conflicts eliminated: 9799 (0,02%) Realigned with forced non-floating bound: 2850810 (6,3%) Realigned with forced non-floating right bound in left read: 1073 (0%) Realigned with forced non-floating left bound in right read: 1073 (0%) TRB chains: 42581901 (100%) TRB non-functional: 1333878 (3,13%) Trimming report: R1 reads trimmed left: 2939 (0,01%) R1 reads trimmed right: 3767 (0,01%) Average R1 nucleotides trimmed left: 4.6647841922360504E-4 Average R1 nucleotides trimmed right: 8.609750214475757E-4 R2 reads trimmed left: 5810 (0,01%) R2 reads trimmed right: 2225 (0%) Average R2 nucleotides trimmed left: 0.005369949631292646 Average R2 nucleotides trimmed right: 0.0012526648411235104 Tag parsing report: Execution time: 0ns Total reads: 45247538 Matched reads: 44307359 (97,92%) Projection +R1 +R2: 44307359 (97,92%) For variant 0: For projection +R1 +R2: UMI:Left position: 17: + 18680889 (42,16%) = 18680889 (42,16%) 18: + 13795379 (31,14%) = 32476268 (73,3%) 19: + 11831091 (26,7%) = 44307359 (100%) UMI:Right position: 31: + 18680889 (42,16%) = 18680889 (42,16%) 32: + 13795379 (31,14%) = 32476268 (73,3%) 33: + 11831091 (26,7%) = 44307359 (100%) R1:Left position: 52: + 18680889 (42,16%) = 18680889 (42,16%) 53: + 13795379 (31,14%) = 32476268 (73,3%) 54: + 11831091 (26,7%) = 44307359 (100%) R1:Right position: 52~148: + 12031255 (27,15%) = 12031255 (27,15%) 149: + 8409710 (18,98%) = 20440965 (46,13%) 150: + 16140918 (36,43%) = 36581883 (82,56%) 151: + 7725476 (17,44%) = 44307359 (100%) R2:Left position: 20 Variants: 0 Cost: 0: + 40947897 (92,42%) = 40947897 (92,42%) 5~10: + 3359462 (7,58%) = 44307359 (100%) UMI length: 14 R1 length: 0~93: + 1566829 (3,54%) = 1566829 (3,54%) 94: + 9478532 (21,39%) = 11045361 (24,93%) 95~96: + 5682711 (12,83%) = 16728072 (37,75%) 97: + 14716785 (33,22%) = 31444857 (70,97%) 98~99: + 12862502 (29,03%) = 44307359 (100%) R2 length: 0~129: + 9874169 (22,29%) = 9874169 (22,29%) 130: + 13121902 (29,62%) = 22996071 (51,9%) 131: + 21311288 (48,1%) = 44307359 (100%)

Analysis date: Mon Jul 31 07:58:23 CEST 2023 Input file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R1.fastq.gz,/Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R2.fastq.gz Output file(s): /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Version: 4.4.1; built=Thu Jul 06 19:36:41 CEST 2023; rev=f7cd556c6c; lib=repseqio.v3.0.1 Command line arguments: align --report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.align.report.txt --json-report /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.align.report.json --preset milab-human-rna-tcr-umi-multiplex /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R1.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T09/D1454T09_trimmed_R2.fastq.gz /Users/alegac/Documents/bulk_tcr/D1454T09/results_09.vdjca Analysis time: 43,96m Total sequencing reads: 45247538 Successfully aligned reads: 42581901 (94,11%) Coverage (percent of successfully aligned): CDR3: 99,87% 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?): 378238 (0,84%) Alignment failed: absence of V hits: 695317 (1,54%) Alignment failed: absence of J hits: 509594 (1,13%) Alignment failed: no target with both V and J alignments: 142309 (0,31%) Alignment failed: absent barcode: 940179 (2,08%) Overlapped: 42961629 (94,95%) Overlapped and aligned: 42216775 (93,3%) Overlapped and not aligned: 744854 (1,65%) Alignment-aided overlaps, percent of overlapped and aligned: 79675 (0,19%) No CDR3 parts alignments, percent of successfully aligned: 182 (0%) Partial aligned reads, percent of successfully aligned: 56224 (0,13%) V gene chimeras: 223 (0%) J gene chimeras: 6777 (0,01%) Paired-end alignment conflicts eliminated: 9799 (0,02%) Realigned with forced non-floating bound: 2850810 (6,3%) Realigned with forced non-floating right bound in left read: 1073 (0%) Realigned with forced non-floating left bound in right read: 1073 (0%) TRB chains: 42581901 (100%) TRB non-functional: 1333878 (3,13%) Trimming report: R1 reads trimmed left: 2939 (0,01%) R1 reads trimmed right: 3767 (0,01%) Average R1 nucleotides trimmed left: 4.6647841922360504E-4 Average R1 nucleotides trimmed right: 8.609750214475757E-4 R2 reads trimmed left: 5810 (0,01%) R2 reads trimmed right: 2225 (0%) Average R2 nucleotides trimmed left: 0.005369949631292646 Average R2 nucleotides trimmed right: 0.0012526648411235104 Tag parsing report: Execution time: 0ns Total reads: 45247538 Matched reads: 44307359 (97,92%) Projection +R1 +R2: 44307359 (97,92%) For variant 0: For projection +R1 +R2: UMI:Left position: 17: + 18680889 (42,16%) = 18680889 (42,16%) 18: + 13795379 (31,14%) = 32476268 (73,3%) 19: + 11831091 (26,7%) = 44307359 (100%) UMI:Right position: 31: + 18680889 (42,16%) = 18680889 (42,16%) 32: + 13795379 (31,14%) = 32476268 (73,3%) 33: + 11831091 (26,7%) = 44307359 (100%) R1:Left position: 52: + 18680889 (42,16%) = 18680889 (42,16%) 53: + 13795379 (31,14%) = 32476268 (73,3%) 54: + 11831091 (26,7%) = 44307359 (100%) R1:Right position: 52~148: + 12031255 (27,15%) = 12031255 (27,15%) 149: + 8409710 (18,98%) = 20440965 (46,13%) 150: + 16140918 (36,43%) = 36581883 (82,56%) 151: + 7725476 (17,44%) = 44307359 (100%) R2:Left position: 20 Variants: 0 Cost: 0: + 40947897 (92,42%) = 40947897 (92,42%) 5~10: + 3359462 (7,58%) = 44307359 (100%) UMI length: 14 R1 length: 0~93: + 1566829 (3,54%) = 1566829 (3,54%) 94: + 9478532 (21,39%) = 11045361 (24,93%) 95~96: + 5682711 (12,83%) = 16728072 (37,75%) 97: + 14716785 (33,22%) = 31444857 (70,97%) 98~99: + 12862502 (29,03%) = 44307359 (100%) R2 length: 0~129: + 9874169 (22,29%) = 9874169 (22,29%) 130: + 13121902 (29,62%) = 22996071 (51,9%) 131: + 21311288 (48,1%) = 44307359 (100%)

mizraelson commented 1 year ago

Duplicate