Package: minorg
MINORgMinimumNon-ReferencegRNA finderFinds the minimum gRNA set required to target multiple alignable genes in multiple non-reference genomesAvailable as both command line application and Python packagePreprint:https://www.biorxiv.org/content/10.1101/2022.03.10.481891AvailabilitySome dependencies are not available for Windows. Windows users should use a Linux emulator to run MINORg.InstallationA version of MINORg is available at test.pypi.org. You may follow thisguideto install MINORg and its dependencies.RequirementsPython 3BLAST+ suiteMAFFTBEDToolsLinksTutorial, example, and documentation:https://rlrq.github.io/MINORgDetailed overview of steps in the programme:https://tinyurl.com/sr84ae9e(Google slides) (not up to date)Flowchart to select & use appropriate input parameters:https://tinyurl.com/jyke76b8(PDF) (not up to date)IMPTPlease refer to slides/PDF in the 'Links' section for execution details for the version on the workstation (accessible only to lab members and guests with accounts).Overview of stepsIdentify candidate targets in non-reference genomeExtract user-specified reference gene(s) from a reference genome (.fasta) using GFF3 annotation (.gff)Sequence(s) include intronsOptional: User may specify a protein domain (using CDD PSSM-ID) to restrict searchCDS-only regions of user-specified reference gene(s) from a reference genome (.fasta) will be extracted and translated using GFF3 annotationRPS-BLAST protein sequence(s) to domain database and identify domain range(s)Extract user-specified reference gene(s) from a reference genome (.fasta) using GFF3 annotation (.gff) and restricted to the corresponding genomic coordinates of the domainsBLASTn reference gene(s) against non-reference genome(s) (.fasta)Filter hits by minimum % identity (optional)Merge overlapping hits within specified distance of each other (to accommodate introns/insertions)Filter merged hits for minimum length and % identity into target sequencesFilter target sequences for those with best alignment to target genes(s) (optional)Ensures that genes that are similar but not part of the set of user-specified target gene(s) will not be targetedIdentify candidate gRNA in non-reference targetsRestricted by user-specified PAM and gRNA lengthScreen candidate gRNAEliminate candidate gRNA with off-target hitsMask targets in non-reference genome(s) (.fasta)Only regions the length of targets with 100% identity to targets will be maskedAll non-reference genomes provided will be screened simultaneously so all candidate gRNA that pass this screening test should not have off-targets in any of the non-reference genomes providedUser may also provide sequences to check againstBLASTn candidate gRNA against masked non-reference and reference genome(s)Optional: Screen reference genome alsoEliminate candidate gRNA with hits outside masked regions in non-reference genome(s) and fail maximum match/gaps criteriaEliminate candidate gRNA that do not align within the CDS of reference genesExtract CDS-only regions of user-specified reference gene(s) from a reference genome (.fasta) using GFF3 annotation (.gff)If the user specified a domain, the range will be restricted accordinglyAlign non-reference target sequences (output of step 1.5) with reference sequences from steps 1.1 (or 1.1.3 if domain is specified) and 2.1For all candidate gRNA, check their position in the alignment (based on where in each non-reference target they originate) and eliminate any gRNA that do not align withinAT LEAST ONEreference gene's desired featureUsers may change the desired feature (default is CDS)Find minimum gRNA set that covers all target sequencesInputsStep 1Data:Reference genome (--ref xxx.fasta)Reference GFF3 annotation (--gff xxx.gff)Non-reference sequences/genome (--nonref xxx.fasta)Parameters:Gene IDs (--gene)Used with:Accession/individual (-i) ORQuery fasta file (-q xxx.fasta)Target sequences (--target xxx.fasta)Optional parameters:Minimum hit % identity (--minid 85 (%))Minimum candidate target length (--minlen 0 (bp))Maximum merge buffer (--buffer 100 (bp))Optional for domain restriction:PSSM-ID (--domain) and rpsblast+ database (--db)Step 2Parameters:PAM (--pam SpCas9)gRNA length (--length 20 (bp))Step 3Optional parameters:Minimum off-target gaps (--ot-gap 0)Minimum off-target mismatch (--ot-mismatch 1 (bp))Optional data:Background sequences (--background xxx.fasta)Step 4Optional paramters:Number of sets to output (--set 1)Manually approve each gRNA set (--manual)