fail in docker: Total 0 white-listed Barcodes

[LinAU]

Thanks for your work.
Everything works well with the demo testing data. However, when I have tested scRNAseq data from 10xgenomics 3' v2. Although the first part runs well. all reads were filtered out due to no white listed BC as lalevin.log file below:

[2024-03-18 20:40:50.427] [alevinLog] [info] Found 70629 transcripts(+0 decoys, +0 short and +0 duplicate names in the index)
[2024-03-18 20:40:50.466] [alevinLog] [info] Filled with 70629 txp to gene entries
[2024-03-18 20:40:50.472] [alevinLog] [info] Found all transcripts to gene mappings
[2024-03-18 20:40:50.479] [alevinLog] [info] Processing barcodes files (if Present)

[2024-03-18 20:54:52.178] [alevinLog] [info] Done barcode density calculation.
[2024-03-18 20:54:52.178] [alevinLog] [info] # Barcodes Used: �[32m316403175�[0m / �[31m316403175�[0m.
[2024-03-18 20:54:52.178] [alevinLog] [info] Done importing white-list Barcodes
[2024-03-18 20:54:52.178] [alevinLog] [warning] Skipping 200 Barcodes as no read was mapped
[2024-03-18 20:54:52.178] [alevinLog] [info] Total 0 white-listed Barcodes
[2024-03-18 20:54:52.178] [alevinLog] [info] Sorting and dumping raw barcodes
[2024-03-18 20:54:58.507] [alevinLog] [warning] Total 100% reads will be thrown away because of noisy Cellular barcodes.
[2024-03-18 20:54:58.507] [alevinLog] [info] Done populating Z matrix
[2024-03-18 20:54:58.507] [alevinLog] [warning] 0 Whitelisted Barcodes with 0 frequency
[2024-03-18 20:54:58.507] [alevinLog] [info] Total 0 CB got sequence corrected
[2024-03-18 20:54:58.507] [alevinLog] [info] Done indexing Barcodes
[2024-03-18 20:54:58.507] [alevinLog] [info] Total Unique barcodes found: 892257
[2024-03-18 20:54:58.507] [alevinLog] [info] Used Barcodes except Whitelist: 0
I used the whitelist file from cellranger, and here it is my docker parameters:
#!/bin/bash

main parameters

INPUT="/mnt/d/data_analysis/Analysis_2023_NAFLD_sc/9.isoform.analysis_v2/5.NAFLD.docker/1.fq/GSM4041150"
OUTPUT="/mnt/d/data_analysis/Analysis_2023_NAFLD_sc/9.isoform.analysis_v2/4.test.docker/ScasaOut_v10"
ref="/mnt/d/data_analysis/Analysis_2023_NAFLD_sc/9.isoform.analysis_v2/4.test.docker/refMrna.fa.gz"
index="YES" #when index="YES", scasa will index the reference fasta file and write in index_dir. This index_dir cam be reused for other run
#index_dir="/path/to/PreBuilt_REF_INDEX" #when index="NO", scasa will use directly the reference indexing in index_dir
nthreads=12
tech="10xv2"
whitelist="/mnt/d/data_analysis/Analysis_2023_NAFLD_sc/9.isoform.analysis_v2/4.test.docker/Test_Dataset/737K-august-2016.txt"
cellthreshold="none"
project="My_Project"

other parameters

samplesheet="NULL"
mapper="salmon_alevin"
xmatrix="alevin"
postalign_dir=""
createxmatrix="NO"

May I ask for your help? Thanks a lot!
KR
Lin

[LinAU]

BTW, I have checked the top10 BC in raw_cb_frequency.txt. I could find them all in my whitelist file manually.

KR,
Lin

[LinAU]

Here are the top BC in raw_cb_frequency.txt in folder SCASA_My_Project_20240318203735\1ALIGN\Sample_S1_L001_alignout\alevin

CTGTGCTTCGCCAGCA 1252648
AGTGTCAAGCAGCCTC 1155715
ACTGAGTCAATGGTCT 1036019
ATTGGACAGTTAACGA 1009156
GGCTCGACAGCCTATA 908019
GGGACCTCAATGGAAT 887484
TTCCCAGTCTTCAACT 788127
TGCCCTATCAGCCTAA 757341
CTCAGAATCATCGCTC 696810
GTAGGCCGTCCATGAT 690158
CAGGTGCAGACAAGCC 681130
CAGAATCTCGTCCAGG 675500

[nghiavtr]

Hi @LinAU ,

Thank you for using Scasa.
From your log file and information you provide, it is hard to know the exact issue.
Could you send us a sample of your data which produced the issue? Then we can investigate the issue in details.

Best,
Nghia

[LinAU]

Dear Nghia, Thanks! How could I share the the fq file? Is it OK I share with Onedrive? KR Lin

…

________________________________ 发件人: Trung Nghia Vu ***@***.***> 发送时间: Thursday, March 21, 2024 8:18:52 PM 收件人: eudoraleer/scasa ***@***.***> 抄送: Lin Lin ***@***.***>; Mention ***@***.***> 主题: Re: [eudoraleer/scasa] fail in docker: Total 0 white-listed Barcodes (Issue #11) Hi @LinAU<https://github.com/LinAU> , Thank you for using Scasa. From your log file and information you provide, it is hard to know the exact issue. Could you send us a sample of your data which produced the issue? Then we can investigate the issue in details. Best, Nghia ― Reply to this email directly, view it on GitHub<#11 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AJNL46MHC2RZXJKNAMQ2FDDYZMXBZAVCNFSM6AAAAABE4K2NKWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDAMJTGQYDQNBXGE>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[nghiavtr]

How big the files are? If they are not too big, Onedrive is ok for me.
Please send the link via my email

Nghia

[LinAU]

Dear Nghia, Thanks! I have shared it with you via Onedrive. KR, Lin From: Trung Nghia Vu ***@***.***> Sent: Friday, 22 March 2024 8:52 AM To: eudoraleer/scasa ***@***.***> Cc: Lin Lin ***@***.***>; Mention ***@***.***> Subject: Re: [eudoraleer/scasa] fail in docker: Total 0 white-listed Barcodes (Issue #11) How big the files are? If they are not too big, Onedrive is ok for me. Please send the link via my email: ***@***.******@***.***> Nghia — Reply to this email directly, view it on GitHub<#11 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AJNL46O2FRAR5TUC5H5D46DYZPPIXAVCNFSM6AAAAABE4K2NKWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDAMJUGUZDQMZWHA>. You are receiving this because you were mentioned.Message ID: ***@***.******@***.***>>

[nghiavtr]

Tks, I have been able to access the files. I will get back to you as soon as I have some news

N

[nghiavtr]

Hi @LinAU
I have carefully investigated the issue with your input files. Later on I found that the issue was not from Scasa but raised by Alevin.

The problem is that Alevin does not work for the whitelist from 737K-august-2016.txt, as it describes in their webpage (https://salmon.readthedocs.io/en/latest/alevin.html#whitelist):

Not 10x 737k whitelist
This flag does not use the technologically defined whitelisted cellular barcodes provided by 10x, instead it’s a per experiment level list of subsampled cellular barcodes that need to quantified for consistency with other tools for example an input would be a file generated by cellranger with the name barcodes.tsv (uncompressed).

So, following the instruction, you might run cellranger first with your data to get barcodes.tsv, then use it as the input for the whitelist.

Best,
Nghia

[LinAU]

Thank you very much! I will test it. KR, Lin From: Trung Nghia Vu ***@***.***> Sent: Tuesday, 26 March 2024 8:42 PM To: eudoraleer/scasa ***@***.***> Cc: Lin Lin ***@***.***>; Mention ***@***.***> Subject: Re: [eudoraleer/scasa] fail in docker: Total 0 white-listed Barcodes (Issue #11) Hi @LinAU<https://github.com/LinAU> I have carefully investigated the issue with your input files. Later on I found that the issue was not from Scasa but raised by Alevin. The problem is that Alevin does not work for the whitelist from 737K-august-2016.txt, as it describes in their webpage (https://salmon.readthedocs.io/en/latest/alevin.html#whitelist): Not 10x 737k whitelist This flag does not use the technologically defined whitelisted cellular barcodes provided by 10x, instead it's a per experiment level list of subsampled cellular barcodes that need to quantified for consistency with other tools for example an input would be a file generated by cellranger with the name barcodes.tsv (uncompressed). So, following the instruction, you might run cellranger first with your data to get barcodes.tsv, then use it as the input for the whitelist. Best, Nghia - Reply to this email directly, view it on GitHub<#11 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AJNL46PI2Y3MSR3KIO7IJF3Y2G6ODAVCNFSM6AAAAABE4K2NKWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDAMRRGMZDEMZZGI>. You are receiving this because you were mentioned.Message ID: ***@***.******@***.***>>