bretonics/chomp

Search guide-RNA sequences given a genome or gene sequence

CHOMP 🐊

CRISPR Tool

CHOMP will search for all 'n' window sized guide RNAs (gRNAs) sequences containing an NGG at the tail end. Default window size is 23.

ex.) ATGTAGCTAGCTAGCTAGTAGGG.

It will report how many occurrences of this sequence are present in the target sequence (off-target sites), along with number of matching bases for each subject/query hit. You can use that to determine which gRNAs you may want to use as CRISPR target(s).

Run CHOMP

perl chomp.pl -seq usr/test.fasta -out gRNAs

Arguments

-seq                Sequence file to search gRNAs [required]
-genome             Genome sequence file(s) to BLAST search (search instead of -seq)
-down               Down sequence to append
-up                 Up sequence to append
-window             Window size for gRNA oligo (default = 23)
-ss                 Secondary structure prediction
-out                Out file name [required]
-outdir             Out directory name
-help               Shows this message

Output

Writes 2 files under the default directory gRNAs:

'.fasta'

Fasta file of each gRNA sequence found.

>gRNA_0:0
ATGTAGCTAGCTAGCTAGTAGGG
>gRNA_1:23
AAAAAATTTTCTCTATCTAACGG
>gRNA_2:24
AAAAATTTTCTCTATCTAACGGG
>gRNA_3:115
TGTGATCACGTACTATTATGCGG
>gRNA_4:149
AAAAATCCCATCGATCTAGCAGG
>gRNA_5:154
TCCCATCGATCTAGCAGGCCCGG
.
.
.
>gRNA_16:99
ATAGTACGTGATCACAGTCATGG

Suffix digit after ':' denotes nucleotide position in sequence where gRNA was found. Ex.) gRNA_16:99 , gRNA was found starting at nucleotide position 99 in -seq sequence.

'.txt'

Report with each gRNA sequence's details.

CHOMP will report how many occurrences of this sequence are present in the target sequence (off-target sites), along with the number of base pair matches (identities) for each. You can use this to determine which gRNA sequence is best to use for target.

Name	Sequence	Strand	Palindrome	Subject	Start	Occurrences	Identities
gRNA_13	TCGTCATGCATGCTCGCTCCGGG	reverse	No	test	173	1	8
gRNA_12	TTCGTCATGCATGCTCGCTCCGG	reverse	No	test	174	1	8
gRNA_3	TGTGATCACGTACTATTATGCGG	plus	No	test	116	2	8
gRNA_16	ATAGTACGTGATCACAGTCATGG	reverse	No	test	109	2	8
gRNA_8	AAAAAAAATTTTCCCTATCGGGG	plus	No	test	195	1	9
gRNA_7	GAAAAAAAATTTTCCCTATCGGG	plus	No	test	194	1	9
gRNA_6	CGAAAAAAAATTTTCCCTATCGG	plus	No	test	193	1	9
gRNA_9	AAAAAAATTTTCCCTATCGGGGG	plus	No	test	196	1	9
gRNA_4	AAAAATCCCATCGATCTAGCAGG	plus	No	test	150	6	11,9,7
gRNA_0	ATGTAGCTAGCTAGCTAGTAGGG	plus	No	test	1	4	14,12,10
gRNA_2	AAAAATTTTCTCTATCTAACGGG	plus	No	test	25	3	15,10,8
gRNA_15	TCCGGGCCTGCTAGATCGATGGG	reverse	No	test	156	6	15,9,7
gRNA_14	CTCCGGGCCTGCTAGATCGATGG	reverse	No	test	157	6	15,9,7
gRNA_5	TCCCATCGATCTAGCAGGCCCGG	plus	No	test	155	6	15,9,7
gRNA_1	AAAAAATTTTCTCTATCTAACGG	plus	No	test	24	3	16,10,8
gRNA_11	TATAGCATGGGCCCCCGATAGGG	reverse	No	test	207	1	23
gRNA_10	CTATAGCATGGGCCCCCGATAGG	reverse	No	test	208	1	23

Table is sorted in increasing order using the top identity for each gRNA sequence, and then sorted by number of occurrences, in current subject.

chomp's Issues

BLAST against multiple sequences

Add support to BLAST against multiple chromosomes or sequences.

• append name (-out and subject sequence) to blast.html output. Ex.) blast_geneX_ChrX.html
• mv output to CRISPRS/blast/
• support directory vs. files

Output directory

Fix output directory to a be under default top level gRNA directory

gRNA
    |_gene1
    |_gene2
    |_gene3

Sort based on number or occurrences first

The number of off-target sites should be minimized, thus the number of occurrences (number of times the CRISPR oligo matches back to the sequence -- may it be genomic or onto itself--) the least number of off-target matches the better.

Subroutines

Subroutines:

• setParameters (manage #3)
• getSequence subroutine to return sequence details with BioPerl

Handle process if gene present in same genome searched

In cases where crispr sequences are being searched in sequence-seq, where the same gene sequence is present in the genome -genome, output should reflect handling exact (23 bp) matches.

Since same gene sequence is searched for crisprs, there will be exact matches in the genome for each crispr sequence found. We want to base sorting on 2nd most matched bp in this case and omit exact matches.

"No hits found" in blast search breaks hash sorting

When a crispr sequence has no matching sequences in search sequence, then nothing is stored in hash. This causes an "undefined value" error when sorting unique identities for each match.

• The integrity of the 'targets' hash must be checked to include all crispr occurrences (keys)
• If no matches found for crispr, then store appropriate info in attributes

Create report

Report should have headers with descriptive comments on how the results were generated.

ADD

• Add call parameters
• Time/date
• Number of hits per subject
• Total number of (no)/hits

Multi FASTA

Allow multi fasta file for -genome option.

Currently searches against multiple files passed in -genome option, but only searches first sequence in multi fasta file (or first '>').

ViennaRNA support

Add support for ViennaRNA secondary structure prediction.

STAR Aligner

Possibly switch to STAR RNA-seq aligner to search sequence to CRISPR sequence matches.

Search reverse input sequence

Search CRISPR targets in reverse input sequence.

Arguments to allow setting sorting options

Allow the sorting to be increasing or decreasing of occurrences/identities. This can be achieved in 3 different ways:

• -sorting option setting each or both simultaneously
• -occ option to allow sorting by occurrences to be inc/dec
• -ident option to allow sorting by identities to be inc/dec

CRISPRS sequences differ between files

Different CRISPR sequences in BLAST results than from 'crisprs.txt' file containing detailed information.

• ✔︎ FASTA file names matche with HTML output
• ❌ BLAST output does not match 'crisprs.txt'

Rank off targets

Get match hits for each CRISPR and rank matches according to how many bp are matched. Not only how many occurrences per target throughout genome but which has the least matching base pairs.

Number of occurrences incorrect

The number of occurrences per CRISPR query match is incorrect. Each successive match gets it's previous match added to the number of occurrences.

This array is not flushing.

Seems to work without -genomes option...what don't I see

Sorting using 'No hits' gets messed up

After fixing issue #20, the removal of CRISPRs with 'No Hits' results in removing them completely when sorting on line 337.

#20 fixes 'undef' issue, but introduces bug where CRISPR may not necessarily be missing in all subject sequences (not only from the singular subject where it is not found). Currently it completely omits those others.

Searching CRISPR targets against subject_1
CRISPR_22 has 0 hits here
CRISPR_50 has 0 hits here
CRISPR_72 has 0 hits here
CRISPR_93 has 0 hits here
CRISPR_129 has 0 hits here

Searching CRISPR targets against subject_2
CRISPR_25 has 0 hits here
CRISPR_91 has 0 hits here

So instead of omitting CRISPRS 22, 50, 72, 93, 129 only for subject_1, it does it for all subjects.

If 'No Hits' CRISPRs are not removed, sorting fails. Conundrum.

Refactor sequence handling

Handling of sequence information should reside in module and check for errors:

• mv getSeqDetails to Search module
• croak if sequence empty

Add genome parameter

Add the following parameters as command line arguments:

• -genome: Genome file to use in blast search instead of self -seq option

blast subroutine - BAD data structure

Anonymous hash data structure incorrectly constructed at line 54.

May be related to #6 , but needs to correctly pair key->value

'CRISPR_9' => [
                          {
                            'qseqid' => '1',
                            'qend' => '25',
                            'qstart' => '23',
                            'nident' => undef,
                            'pident' => '23
',
                            'sstart' => '47',
                            'send' => 'plus',
                            'sstrand' => '100.000'
                          },
                          {
                            'pident' => '15
',
                            'sstart' => '213',
                            'send' => 'plus',
                            'sstrand' => '93.750',
                            'nident' => undef,
                            'qseqid' => '1',
                            'qstart' => '16',
                            'qend' => '198'
                          },