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License: MIT License
Bowtie-based alignment of CRISPR gRNA spacer sequences
License: MIT License
Hi Jean-Philippe,
thanks for this great R package.
I would like to use crisprVerse package to design gRNAs in plant species and speed up the CRISPR construct design.
I have tested the package with the new sorghum genome (JGIv5) but I think something went wrong with the function addSpacerAlignmentsIterative
In particular, I do see differences in the results when I run the functions addSpacerAlignmentsIterative and runCrisprBowtie .
Although the parameter I used were the same ( bowtie, # mismatches, the two functions reported different results for the 21 spacers found :
Please see output below.
In addition, only few alignments derived from addSpacerAlignmentsIterative were properly stored in the S4 object guideSet:
For reproducibility, I have created a google drive folder with the data used and the R code to run the analysis:
https://drive.google.com/drive/folders/1SP7ijDdpKqB1ElWSpfAyewIsM0CJm2lJ?usp=share_link
#These are the outputs for a quick look:
addSpacerAlignmentsIterative(guideSet,
aligner_index="Documents/Ref_sorghum/v5.1/assembly/Sbicolor_730_v5.0", #change the path to the index according to your local DIR
aligner="bowtie",
bsgenome=bsgenome,
n_mismatches=3,
both_strands = TRUE,
canonical = FALSE)
[runCrisprBowtie] Using BSgenome.Sbicolor.JGI.sorBic5
[runCrisprBowtie] Searching for SpCas9 protospacers
reads processed: 21
reads with at least one alignment: 21 (100.00%)
reads that failed to align: 0 (0.00%)
Reported 21 alignments
[runCrisprBowtie] Using BSgenome.Sbicolor.JGI.sorBic5
[runCrisprBowtie] Searching for SpCas9 protospacers
reads processed: 21
reads with at least one alignment: 21 (100.00%)
reads that failed to align: 0 (0.00%)
Reported 22 alignments
[runCrisprBowtie] Using BSgenome.Sbicolor.JGI.sorBic5
[runCrisprBowtie] Searching for SpCas9 protospacers
reads processed: 21
reads with at least one alignment: 21 (100.00%)
reads that failed to align: 0 (0.00%)
Reported 75 alignments
[runCrisprBowtie] Using BSgenome.Sbicolor.JGI.sorBic5
[runCrisprBowtie] Searching for SpCas9 protospacers
reads processed: 21
reads with at least one alignment: 21 (100.00%)
reads that failed to align: 0 (0.00%)
Reported 1384 alignments
crisprNuclease <- SpCas9
spacers <- as.character(protospacers(guideSet))
runCrisprBowtie(spacers,
crisprNuclease=crisprNuclease,
n_mismatches=3,
canonical=FALSE,
all_alignments = TRUE,
bowtie_index="Documents/Ref_sorghum/v5.1/assembly/Sbicolor_730_v5.0") #change the path to the index according to your local DIR
[runCrisprBowtie] Searching for SpCas9 protospacers
reads processed: 84
reads with at least one alignment: 84 (100.00%)
reads that failed to align: 0 (0.00%)
Reported 178 alignments
Thanks,
Edo
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