CFBI

CFBI (Calculating Frequencies of Base-substitutions and Indels) is the custome Perl and Shell scripts for calculating frequencies of base subsutitution and indels.

Version: 1.0.0

Features

Calculate base substitution and indel frequencies of target gene for CRISPR genome editing.

BAM format file

BAM file was originally mapped form BWA-MEM.

Prerequisites

Software / Package

• Perl v5.26.2
• BioPerl v1.7.2
• BWA Version: 0.7.17
• SAMtools Version: 1.9
• GCC
• GNU coreutils

Usage:

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To calculate base substitution and indel frequencies, BAM format file was generated firstly.

All Perl and Shell scripts were marked 'bold italic'.

• 1. Please export the CFBI directory and BioPerl to your $PATH.

export PATH="~/CFBI-master:$PATH";
export PATH="~/Bio:$PATH";

• 2. BWA index target sequences. '01_bwa_mem_index.sh'.

sh 01_bwa_mem_index.sh target.fa

• 3. BWA-MEM mapping with DNA-seq reads (FASTQ). For paired-end sequencing, only R1 reads were used. '02_bwa_mem_mapping.sh'.

sh 02_bwa_mem_mapping.sh target.fa sample_R1.fq sample_R1

• 4. Calculate base substitution with mapped reads (BAM). '03_base_substitution.sh'.

sh 03_base_substitution.sh sample_R1.bam target.fa sample_R1

Output file sample_R1.xls is an example result of base substitution.

• 5. Calculate indel frequencies with target gene InDel location. '04_indel_frequencies.sh'.

sh 04_indel_frequencies.sh sample_R1.bam EXM1 250 300 ascii.txt

Output file EMX1_indel_frequencies.txt is an example for the number of indel frequencies of target EMX1.

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Input files

1. Target sequences with FASTQ format. (target.fa)
2. DNA-seq R1 reads with FASTQ format. (sample_R1.fq)
3. Name of BAM file. (sample_R1)
4. Target gene symbol. (EMX1)
5. Start location for indel frequencies calculation of target gene. (250, cutting site -25 bp for EMX1)
6. End location for indel frequencies calculation of target gene. (300, cutting site +25 bp for EMX1)
7. The sequence quality of reads by ASCII code. ascii.txt

Output files

See details in sample_R1.xls, the example output file for base substitution.

Field	Description
Gene symbol	Name of gene
Mut location	Location of mutant site
Ref base	Sequence of base
Flanking base	± 1 bp flanking sequence of base
# of mapped reads	Number of mapped reads for target gene
# of total mutant reads	Number of total mutant reads for target gene
% of total mutant reads	Ratio of total mutant reads for target gene
# of mutant A	Number of reads for mutant A
% of mutant A	Ratio of reads for mutant A
# of mutant C	Number of reads for mutant C
% of mutant C	Ratio of reads for mutant C
# of mutant G	Number of reads for mutant G
% of mutant G	Ratio of reads for mutant G
# of mutant N	Number of reads for mutant N
% of mutant N	Ratio of reads for mutant N
# of mutant T	Number of reads for mutant T
% of mutant T	Ratio of reads for mutant T

See details in EMX1_indel_frequencies.txt, the example output file for indel frequencies.

Field	Description
Gene symbol	Name of gene
Type of reads	Type of all reads or indel reads
# of reads	Number of all reads or indel reads

Citation

The related paper about CFBI is submitted.

License

Copyright (C) 2021 YangLab. Licensed GPLv3 for open source use or contact YangLab ([email protected]) for commercial use.