Trinity/Igblast failure

[mbartl13]

Hi-

I am getting an error that I am not sure how to address. I think igblast is failing because trinity failed? But the install looks ok to me. Any suggestions for how to fix?

./BALDR/BALDR --paired /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R1_001.fastq.gz,/dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R2_001.fastq.gz --trimmomatic /jhpce/shared/jhpce/modulefiles/libd/software/Trimmomatic2/Trimmomatic-0.32/trimmomatic-0.32.jar --adapter ./software/Trimmomatic2/Trimmomatic-0.32/adapters/NexteraPE-PE.fa --BALDR /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR --trinity /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity --memory 64G --threads 32 --STAR /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/STAR_MacaM/STAR --STAR_index /genome --igblastn /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/igblast_1.16.0/ncbi-igblast-1.16.0/bin/igblastn Error: 2021-08-25 13:00:22 Skipping Trimmomatic. Found Trimmed files 2021-08-25 13:00:22 Skipping STAR. Found bam file 2021-08-25 13:00:22 Skipping extracting reads. Found listreads file in IG-mapped/IG_reads 2021-08-25 13:00:22 Skipping extracting IG fastq file. Found fastq.gz file in IG-mapped/IG_fastq 2021-08-25 13:00:22 Skipping merging IG & Unmapped reads. Found fastq.gz file in IG-mapped_Unmapped/IG-mapped_Unmapped_fastq 2021-08-25 13:00:22 Running Trinity assembly for --left IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_1P.IG_Unmapped.fastq.gz --right IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_2P.IG_Unmapped.fastq.gz. /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity --seqType fq --full_cleanup --max_memory 64G --left IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_1P.IG_Unmapped.fastq.gz --right IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_2P.IG_Unmapped.fastq.gz --CPU 32 --no_normalize_reads --output IG-mapped_Unmapped/Trinity/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity > IG-mapped_Unmapped/Trinity/Log/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity_log This Perl not built to support threads Compilation failed in require at /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity line 5. BEGIN failed--compilation aborted at /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity line 5. 2021-08-25 13:00:22 Finished Trinity assembly. Assembled reads written in IG-mapped_Unmapped/Trinity. /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/igblast_1.16.0/ncbi-igblast-1.16.0/bin/igblastn -germline_db_V /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_V -germline_db_J /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_J -germline_db_D /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_D -organism human -domain_system imgt -query IG-mapped_Unmapped/Trinity/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity.Trinity.fasta -auxiliary_data /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/optional_file/human_gl.aux -show_translation -out IG-mapped_Unmapped/IgBLAST/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.blastout -num_threads 32 -db /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_C -evalue 0.001 -outfmt 7 > IG-mapped_Unmapped/IgBLAST/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.blastout.log /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/igblast_1.16.0/ncbi-igblast-1.16.0/bin/igblastn: error while loading shared libraries: libuv.so.1: cannot open shared object file: No such file or directory Cannot open IG-mapped_Unmapped/Trinity/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity.Trinity.fasta at /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/lib/parse_igblast.pl line 27. bowtie2-build IG-mapped_Unmapped/Quantification/full/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.full.fa IG-mapped_Unmapped/Quantification/full/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped Settings: Output files: "IG-mapped_Unmapped/Quantification/full/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped..bt2" Line rate: 6 (line is 64 bytes) Lines per side: 1 (side is 64 bytes) Offset rate: 4 (one in 16) FTable chars: 10 Strings: unpacked Max bucket size: default Max bucket size, sqrt multiplier: default Max bucket size, len divisor: 4 Difference-cover sample period: 1024 Endianness: little Actual local endianness: little Sanity checking: disabled Assertions: disabled Random seed: 0 Sizeofs: void:8, int:4, long:8, size_t:8 Input files DNA, FASTA: IG-mapped_Unmapped/Quantification/full/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.full.fa Error: could not open IG-mapped_Unmapped/Quantification/full/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.full.fa Total time for call to driver() for forward index: 00:00:00 Error: Encountered internal Bowtie 2 exception (#1) Command: /jhpce/shared/community/core/bowtie2/2.9.2/bin/bowtie2-build-s --wrapper basic-0 IG-mapped_Unmapped/Quantification/full/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.full.fa IG-mapped_Unmapped/Quantification/full/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped bowtie2-build IG-mapped_Unmapped/Quantification/VDJ/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.VDJ.fa IG-mapped_Unmapped/Quantification/VDJ/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped Settings: Output files: "IG-mapped_Unmapped/Quantification/VDJ/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped..bt2" Line rate: 6 (line is 64 bytes) Lines per side: 1 (side is 64 bytes) Offset rate: 4 (one in 16) FTable chars: 10 Strings: unpacked Max bucket size: default Max bucket size, sqrt multiplier: default Max bucket size, len divisor: 4 Difference-cover sample period: 1024 Endianness: little Actual local endianness: little Sanity checking: disabled Assertions: disabled Random seed: 0 Sizeofs: void:8, int:4, long:8, size_t:8 Input files DNA, FASTA: IG-mapped_Unmapped/Quantification/VDJ/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.VDJ.fa Error: could not open IG-mapped_Unmapped/Quantification/VDJ/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.VDJ.fa Total time for call to driver() for forward index: 00:00:00 Error: Encountered internal Bowtie 2 exception (#1) Command: /jhpce/shared/community/core/bowtie2/2.9.2/bin/bowtie2-build-s --wrapper basic-0 IG-mapped_Unmapped/Quantification/VDJ/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.VDJ.fa IG-mapped_Unmapped/Quantification/VDJ/index/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped IG-mapped_Unmapped/IgBLAST/tabular/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.igblast_tabular not found Results written to IG-mapped_Unmapped/IgBLAST_quant_sorted_filtered/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.igblast_tabular.quant.sorted.IGH.filtered Results written to IG-mapped_Unmapped/IgBLAST_quant_sorted_filtered/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.igblast_tabular.quant.sorted.IGKL.filtered

[amit-upadhyay]

Can you please share the Trnity log file: IG-mapped_Unmapped/Trinity/Log/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity_log?

[mbartl13]

Hi- the file is there but empty? Added space in file so I could upload but originally 0 KB 13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity_log.txt

[amit-upadhyay]

I apologize for the delay. Will it be possible for you to share the input files?

[mbartl13]

To view 13F-BCR_S5_L001_R1_001.fastq(1).gz, sign inhttps://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/_layouts/15/acceptinvite.aspx?invitation=%7B679F836D%2DC1EA%2D4386%2D853B%2D473032A17924%7D&listId=1b342fe9%2D6dfe%2D4555%2Db400%2Dde6dfb4a81e6&itemId=09c23870%2Df1c4%2D48ae%2D9c69%2Dd0d25a02d846 or create an account.

[mbartl13]

To view 13F-BCR_S5_L001_R2_001.fastq.gz, sign inhttps://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/_layouts/15/acceptinvite.aspx?invitation=%7B1987C436%2D7AB3%2D4543%2DB5C3%2D978E80346509%7D&listId=1b342fe9%2D6dfe%2D4555%2Db400%2Dde6dfb4a81e6&itemId=6dc8806f%2D82ae%2D4fab%2Db544%2Db454859953d6 or create an account.

[mbartl13]

Maggie Bartlett has shared OneDrive for Business files with you. To view them, click the links below. https://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R2_001.fastq.gz [https://r1.res.office365.com/owa/prem/images/dc-generic_20.png]https://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R2_001.fastq.gz 13F-BCR_S5_L001_R2_001.fastq.gzhttps://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R2_001.fastq.gz

https://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R1_001.fastq(1).gz [https://r1.res.office365.com/owa/prem/images/dc-generic_20.png]https://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R1_001.fastq(1).gz 13F-BCR_S5_L001_R1_001.fastq(1).gzhttps://livejohnshopkins-my.sharepoint.com/personal/mbartl13_jh_edu/Documents/Attachments/13F-BCR_S5_L001_R1_001.fastq(1).gz

No worries, see attached.

Thanks! Maggie

---

From: Amit Upadhyay @.> Sent: Sunday, September 12, 2021 8:57 AM To: BosingerLab/BALDR @.> Cc: Maggie Bartlett @.>; Author @.> Subject: Re: [BosingerLab/BALDR] Trinity/Igblast failure (#13)

  External Email - Use Caution

I apologize for the delay. Will it be possible for you to share the input files?

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHubhttps://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2FBosingerLab%2FBALDR%2Fissues%2F13%23issuecomment-917631457&data=04%7C01%7Cmbartl13%40jh.edu%7C956d5f5de0374266de1408d975ecf199%7C9fa4f438b1e6473b803f86f8aedf0dec%7C0%7C0%7C637670482807857085%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=c8staqEHcIyr4JM8ZFHXWLi7%2FeVT1y4jNgxs93hOATE%3D&reserved=0, or unsubscribehttps://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FARPWQ5EO75IFWSH2JKDVMKDUBSPVJANCNFSM5CZSJNQA&data=04%7C01%7Cmbartl13%40jh.edu%7C956d5f5de0374266de1408d975ecf199%7C9fa4f438b1e6473b803f86f8aedf0dec%7C0%7C0%7C637670482807867082%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=SKddTtOk8uC%2BLDfwwdbycKN%2FON9HREZDMAtvj3PYC8I%3D&reserved=0. Triage notifications on the go with GitHub Mobile for iOShttps://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fapps.apple.com%2Fapp%2Fapple-store%2Fid1477376905%3Fct%3Dnotification-email%26mt%3D8%26pt%3D524675&data=04%7C01%7Cmbartl13%40jh.edu%7C956d5f5de0374266de1408d975ecf199%7C9fa4f438b1e6473b803f86f8aedf0dec%7C0%7C0%7C637670482807867082%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=aJ7cdzc2ed26WbELqyy7OmbOq9wAxAk8Ctjg0M6fIvs%3D&reserved=0 or Androidhttps://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fplay.google.com%2Fstore%2Fapps%2Fdetails%3Fid%3Dcom.github.android%26referrer%3Dutm_campaign%253Dnotification-email%2526utm_medium%253Demail%2526utm_source%253Dgithub&data=04%7C01%7Cmbartl13%40jh.edu%7C956d5f5de0374266de1408d975ecf199%7C9fa4f438b1e6473b803f86f8aedf0dec%7C0%7C0%7C637670482807877072%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=%2BQM24o5W%2BAMFuKAHss2Q2jtz5dDUWJjhDNmTghTEciA%3D&reserved=0.

[mbartl13]

Hi- still having issues getting results.

./BALDR/BALDR --paired /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R1_001.fastq.gz,/dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R2_001.fastq.gz --trimmomatic /jhpce/shared/jhpce/modulefiles/libd/Trimmomatic2/Trimmomatic-0.36/trimmomatic-0.36.jar --adapter ./software/Trimmomatic2/Trimmomatic-0.36/adapters/NexteraPE-PE.fa --BALDR /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR --trinity /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity --memory 64G --threads 32 --STAR /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/STAR_MacaM/STAR --STAR_index /genome --igblastn /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/Igblast/ncbi-igblast-1.16.0/bin/igblastn

2022-01-12 10:27:57 Skipping Trimmomatic. Found Trimmed files 2022-01-12 10:27:57 Skipping STAR. Found bam file 2022-01-12 10:27:57 Skipping extracting reads. Found listreads file in IG-mapped/IG_reads 2022-01-12 10:27:57 Skipping extracting IG fastq file. Found fastq.gz file in IG-mapped/IG_fastq 2022-01-12 10:27:57 Skipping merging IG & Unmapped reads. Found fastq.gz file in IG-mapped_Unmapped/IG-mapped_Unmapped_fastq 2022-01-12 10:27:57 Running Trinity assembly for --left IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_1P.IG_Unmapped.fastq.gz --right IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_2P.IG_Unmapped.fastq.gz. /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/trinityrnaseq/Trinity --seqType fq --full_cleanup --max_memory 64G --left IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_1P.IG_Unmapped.fastq.gz --right IG-mapped_Unmapped/IG-mapped_Unmapped_fastq/13F-BCR_S5_L001_R1_001_2P.IG_Unmapped.fastq.gz --CPU 32 --no_normalize_reads --output IG-mapped_Unmapped/Trinity/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity > IG-mapped_Unmapped/Trinity/Log/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity_log 2022-01-12 10:27:57 Finished Trinity assembly. Assembled reads written in IG-mapped_Unmapped/Trinity. /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/Igblast/ncbi-igblast-1.16.0/bin/igblastn -germline_db_V /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_V -germline_db_J /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_J -germline_db_D /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_D -organism human -domain_system imgt -query IG-mapped_Unmapped/Trinity/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped_trinity.Trinity.fasta -auxiliary_data /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/optional_file/human_gl.aux -show_translation -out IG-mapped_Unmapped/IgBLAST/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.blastout -num_threads 32 -db /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/resources/IgBLAST_DB/human/human_IG_C -evalue 0.001 -outfmt 7 > IG-mapped_Unmapped/IgBLAST/13F-BCR_S5_L001_R1_001.IG-mapped_Unmapped.blastout.log /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/Igblast/ncbi-igblast-1.16.0/bin/igblastn: error while loading shared libraries: libuv.so.1: cannot open shared object file: No such file or directory

[amit-upadhyay]

It seems like an error related to igblastn. I have not updated the pipeline to work with newer versions of IgBLAST. Can you please try with IgBLAST v1.6.1? You can also try using the docker image.

[mbartl13]

Thanks, I'm new to using docker- we have singularity on the cluster I use. When I pull the docker image and run with singularity I get: singularity run -B /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/STAR_MacaM/STAR:/genome -B /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/:/data -w /data bosingerlab/baldr /home/tools/BALDR-master/BALDR --paired /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R1_001.fastq.gz,/dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/13F_BCR_L001-ds.b7527f09a164490d9bc13af50cbc4ef9/13F-BCR_S5_L001_R2_001.fastq.gz --trimmomatic /home/tools/Trimmomatic-0.38/trimmomatic-0.38.jar --adapter /home/tools/Trimmomatic-0.38/adapters/NexteraPE-PE.fa --BALDR /home/tools/BALDR-master --trinity /home/tools/trinityrnaseq-Trinity-v2.3.2/Trinity --memory 64G --threads 32 --STAR /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/BALDR/STAR_MacaM/STAR --STAR_index /genome --igblastn /dcl02/leased/giffin/MeV_scSeq/20210519_scSeq_43F_13F/Immcantation/software/igblast_1.16.0/ncbi-igblast-1.16.0/bin/igblastn FATAL: could not open image /data: failed to retrieve path for /data: lstat /data: no such file or directory

[amit-upadhyay]

Sorry, I am not familiar with running it on singularity. Is this the only sample you want to run? I cannot access the files that you shared previously. Can you pls share those again? You can also email me at amit.anil.upadhyay@emory.edu.