Error in cb2_count()

[hjoshi-nyscf]

Hi, I am trying CB2 analysis in R to analyze my CRISPR data. The package is installed properly (I guess) and example data runs fine. When I am trying my own data, I can load the files correctly. but I am having trouble running the next steps.

Error: cb2_count <- run_sgrna_quant(FASTA, df_design) Error in data.frame(sgRNA = quant_ret$sgRNA, sequence = quant_ret$sequence) : arguments imply differing number of rows: 201322, 202586

Commands (successful)

FASTA <- "/CB2analysis/CRISPRi_v2_human.trim_1_39_forward.fa"

df_design <- tribble(~group, ~sample_name, "Pre","IMEV0023-Pre_R1_001","Post", "IMEV0023-Post_R1_001","C2","IMEV0023-C2-S1_R1_001","C6","IMEV0023-C6-S4_R1_001",) %>% mutate( fastq_path = glue("{ex_path}/{sample_name}.fastq.gz") )

df_design A tibble: 4 × 3 group sample_name fastq_path

1 Pre IMEV0023-Pre_R1_001 /extdata/Screen23i/IMEV0023-Pre_R1_001.f… 2 Post IMEV0023-Post_R1_001 /extdata/Screen23i/IMEV0023-Post_R1_001.… 3 C2 IMEV0023-C2-S1_R1_001 /extdata/Screen23i/IMEV0023-C2-S1_R1_001… 4 C6 IMEV0023-C6-S4_R1_001 /extdata/Screen23i/IMEV0023-C6-S4_R1_001…

I am using Rstudio: 2022.12.0+353 R 4.2.2

If you can point me to right direction of resolving this, that would be highly appreciated. HJ

[hyunhwan-jeong]

Hello @hjoshi-nyscf, can you send your CRISPRi_v2_human.trim_1_39_forward.fa to jeong.compbio@gmail.com?

Thank you,

Hyun-Hwan Jeong

[hyunhwan-jeong]

Hi @hjoshi-nyscf,

• The guide library length is 38bp, and the current CB2 only supports guide up to ~20bp. A workaround is going to be that strip the suffix - GTTTAAGAGCTAAGCTGGA would allow CB2 can quantify.
• As default, CB2 uses _ as a delimiter of gene symbol and guide name, and using underscores twice every guide name may cause the problem. I will post a solution today or tomorrow regarding it.

Hyun-Hwan Jeong

[hyunhwan-jeong]

@hjoshi-nyscf one of easy workaround is going to be rename the guide name as "genesymbol_guideRNA". Can you try?

Hyun-Hwan Jeong

[hyunhwan-jeong]

@hjoshi-nyscf have you solved the problem?