Comments (5)
hyunhwan-jeong
commented on July 29, 2024
Hi @monovich, CB2 currently only supports single-end FASTQ files. To support your paired-end read files, I may need further information regarding it. Could you provide further details?
Thank you,
Hyun-Hwan Jeong
from cb2.
monovich
commented on July 29, 2024
Certainly. I have 150 bp paired-end reads for my sgRNA abundance quantification, which is probably overkill for 23nt guides, but it was what our sequencing core is now providing by default to standardize library preps. Obviously providing both files to CB2 doesn't work, so I will need to generate some sort of single file input. I could just utilize the reads from R1 if you think that is appropriate, or I could generate an interleaved FASTQ, but I suspect CB2 would see that as simply twice as many reads treating R1 and R2 as unpaired, so that may skew calculated statistics.
Example first 3 reads from my first sample's R1 and R2 file (as you can see they are paired):
==> 3579-AI-1_TTGAACCG-ATAGGATC_S216_R1_001.fastq <==
@A00437:378:H3JYFDSX2:1:1101:5141:1000 1:N:0:TTGAACCG+ATAGGATC
TNGTGGAAAGGACGAAACACCGCTCCGTCCCCTCCTGCCGCGGTATAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAATTCTAGATCTTGA
+
F#FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:,FFFFFFFFFFFFFFFFFFFF:FFFFFF:,FFFFFFFFFFFFFF:FFFFFF,::FFFFFFFF:FFFFFFFF:FFFFF:FFF,FFFFFF:,FFFF,FFF,FFFFFFFF
@A00437:378:H3JYFDSX2:1:1101:8793:1000 1:N:0:TTGAACCG+ATAGGATC
TNGTGGAAAGGACGAAACACCGTTGGAACAAAGAAAACTCCCGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTCGAATTCTAGATCTTGA
+
F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FF:FFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFF
@A00437:378:H3JYFDSX2:1:1101:12897:1000 1:N:0:TTGAACCG+ATAGGATC
TNGTGGAAAGGACGAAACACCGGAACAGGCAGACACATCTCAGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAATTCTAGATCTTGA
==> 3579-AI-1_TTGAACCG-ATAGGATC_S216_R2_001.fastq <==
@A00437:378:H3JYFDSX2:1:1101:5141:1000 2:N:0:TTGAACCG+ATAGGATC
TCTACTATTCTTTCCCCTGCACTGTACCCCCCAATCCCCCCTTTTCTGTTAAAATTGTGGATGAATACTGCCATTTGTCTCAAGATCTAGAATTCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF:F,FFFFFFFFF:FFFFFFFF:F:FFF,FFFFF,FF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFF:FFF
@A00437:378:H3JYFDSX2:1:1101:8793:1000 2:N:0:TTGAACCG+ATAGGATC
TCTACTATTCTTTCCCCTGCACTGTACCCCCCAATCCCCCCTTTTCTTTTAAAATTGTGGATGAATACTGCCATTTGTCTCAAGATCTAGAATTCGAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F:FFFFF,FFFF:FFFFFFFF:FFFFFFFF::FFFFFF:FFF:,FFFFFFFFFFFFFFFFFF:FFF:F:FFFFFF:FFFFFFFF,FF::F:FFFFFFFFFFFF::FFFF
@A00437:378:H3JYFDSX2:1:1101:12897:1000 2:N:0:TTGAACCG+ATAGGATC
TCTACTATTCTTTCCCCTGCACTGTACCCCCCAATCCCCCCTTTTCTTTTAAAATTGTGGATGAATACTGCCATTTGTCTCAAGATCTAGAATTCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATT
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hyunhwan-jeong
commented on July 29, 2024
Based on my experience, every guide would be located at a constant location (of course, it can be a bit staggered). So, I guess most guides would appear at one of the ends. In other words, if you see a lot of guides in R1, you may not need to use R2. Have you checked the mappability of each R1 and R2? It helps my assumption is correct or not. Otherwise, it will be time to find plan B.
Hyun-Hwan Jeong
from cb2.
monovich
commented on July 29, 2024
That makes good sense. I just quickly ran my R1 file from above on its own through bwa and it looks like I'm getting 99% alignment, so I'll probably just go ahead and use my R1 files. Thanks for the clarification.
from cb2.
hyunhwan-jeong
commented on July 29, 2024
No problem, I think we can close the issue.
from cb2.
Related Issues (20)
- Problem with gene-level statistic HOT 1
- Error in arising in run_sgrna_quant HOT 4
- Cluster setup failed. 31 of 31 workers failed to connect. HOT 2
- run_sgrna_quant report the wrong sequences associated to sgRNA names HOT 2
- maximum guide length HOT 2
- Error in cb2_count() HOT 4
- Is it possible to analyze by inserting one mismatch guide RNA barcode? HOT 3
- Error in measure_gene_stats(sgrna_stat) HOT 3
- Interaction Terms and Complex Designs HOT 1
- Fasta file issue HOT 16
- CB2 vhat estimation with variable total read count
- Choice of count normalisation
- run_sgrna_quant fails HOT 3
- inconsistent between .fq.gz and .fastq? HOT 7
- calc_mappability HOT 1
- export join_count_and_design HOT 2
- plot_count_distribution add export option HOT 1
- gene_stat cpms HOT 2
- Clarity around logFC for gene_stats HOT 3
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