AFSM_seq

Reference based analysis
1.Filter the raw reads obtained from Illumina Raw Data (paired-ends)according to your desired stringency, to produce a set of high-quality (HQ) sequences in fastq format.
2.Lignment reads (HQ, Fastq format) against Barcode file (Fasta format) using scanAP program.
3.Trim barcodes and filter paired-ends using trim_seq.pl. Classified paired-ends using fltfastq2pe.pl. to produce output “R1.trim.pair.fastq” and “R2.trim.pair.fastq”.
4.The filtered sequence reads were aligned to the reference genome using the Bowtie2, allowing a maximum of four mismatches and one gap of up to 3 bp. Using the SAMtools pileup command the variable positions (SNPs) were determined.
5.AFSM_meth_V_1.0.pl are used for analyses of AFSM methylation results were based on comparisons of assembled sequences .
perl AFSM_meth_V_1.0.pl -i output.sort.bam -o output.meth.matrix