XinYang6699 / Methpup

Methpup (METHylation site Picking Up Pipeline) is a pipeline to pick up methylation sites from bisulphite sequnceing reads with known methylation site positions. It takes the raw pair-end Bi-seq data as input, does some quality control and aligns reads to targeted regions using the ultra high-throughput short read aligner Bowtie2, and then analyzes the mapping results, adjusts the reads with insertion/deletion and pick up the methylation sites.

For instructions on using the perl implementation of this software please see README.perl file.

This software is developed by Xin Yang (xin.yang@cimr.cam.ac.uk) in Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory (JDRF/WT DIL). A paper describing this software is in preparing for Genome Biolofy software.


===================== 
Pre-requirement:
=====================

1) Install fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and set the option --fastqcdir to your path to fastqc directory. 
2) Install trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic) and set the option --trimmomatic to your path to trimomatic.jar file.
3) Install cutadapt (https://code.google.com/p/cutadapt/) and set the option --cutadapt to your path to your cutadapt/bin directory.
4) flash (http://ccb.jhu.edu/software/FLASH/) and put it in your $PATH.
5) Install bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) and put it in your $PATH.
6) Python and java are needed to support this software.
7) Copy indel.py and methsite.py under this directory to your $PATH.

====================
Synopsis
====================

./Methpup -i input -o output -f linkseq1 -r linkseq2 -l linklen -t path/to/trimmomatic.jar --refdir=/your-path-to-ref-directory/ -x ref -s siteref --fastqcdir=/your-path-to-fastqc/ --cutadaptdir=/your-path-to-cutadapt/ [--queue=/your-queue-file/] [--phred=33] [--formin=5] [--revmin=4] [-m10] [-M222]

====================
Description
====================

The options for the program as as follows:
    
    -i --indir      The directory saves all input raw fastq files.
    
    -o --outdir     Create all output files in the specified output directory.
                    Please note that this directory must exist as the program
                    will not create it.
 
    -q --queue	    Customized bash files to submit parallele jobs in q. If 
       		    not supplied, the jobs would run locally in current terminal.
		    
    --phred         Input qualities are ASCII chars equal to the Phred quality 
                    plus 33 (by default). This is also called the "Phred+33" encoding,
                    which is used by the very latest Illumina pipelines.

    -f --forseq     The linker sequence that links barcode/adapter and target region in
       		    the 5'end.

    -r --revseq     The linker sequence that links target region and adapter in
       		    the 3'end.

    -l --seqlen     The length of the linker sequence

    --formin        Minimum overlap length between the read and the linker in 
    		    forward reads. If the overlap is shorter than LENGTH, the
		    read is not modified. This reduces the number of bases 
		    trimmed purely due to short random adapter matches (default:
                    5)
    --revmin        Minimum overlap length between the read and the linker in 
    		    forward reads.(default:4)

    -t --trimmomatic
	            The path to trimmomatic.jar file

    -m --min-len    The minimum required overlap length between two
                    reads to provide a confident overlap in stitching. (default:
                    10)

    -M --max-len    Maximum overlap length expected in approximately 90% of 
       		    read pairs in stitching. (default:222)
		      

    -x --ref        a fasta file containing reference sequence

    --refdir        the path to the directory that contain the reference sequence file

    -s --siteref    a tab separated file listing methylation site position 
       		    with one line one region in the following format:
		    gene1(region1)    position1	position2.....
		    gene2(region2)    position1 position2.....
 		    The first base in target region is position 1.

    --fastqcdir     the path to the directory that contain fastqc command

    --cutadaptdir   the path to the directory that contain cutadapt command

    -h              print this usage page


====================
Test
===================

There are several pair-end raw read files under test directory. The output results of this software are saved under test_result directory. To generate this result, you could try this command under the current directory in bash:

./Methpup -i test -o test_result -f GGTCATAGCTGTTTCCTG -r ACTGGCCGTCGTTTTACA -l 18 -t /path-to-trimmomatic.jar/ --refdir=test_result/ref -x myref.fa -s test_result/total_CpG_sites.txt --fastqcdir=/path-to-fastqc/ --cutadaptdir=/path-to-cutadapt/


====================
BUGS
====================
Any bugs in Methpup should be reported to xin.yang@cimr.cam.ac.uk


====================
Acknowledgement
====================
I'd like to thank Dr Chris Wallace for her supervision on this work.