riceBS

Rice Bisulfide sequencing support

I. Prepare your files for Bisulfite Bioinformatic processing

1. Start with making a directory called "reads" and download fastq files into that directory.

2. create a file with old_names new_names for fastq

ex:

%] cat Bio20_2014_Sprint_run3 flowcell246_lane1_pair1_GGCTAC.fastq A123_1.p1.fq flowcell246_lane1_pair2_GGCTAC.fastq A123_1.p2.fq flowcell246_lane1_pair1_ATGTCA.fastq NB_1.p1.fq flowcell246_lane1_pair2_ATGTCA.fastq NB_1.p2.fq

3. rename (really these are symlinked files) the files with informative names. Use rename_fq.pl with 2 arguments a. the directory of the files b. the file made in step 1.

ex:

%]rename_fq.pl ~/bigdata/Bio20_2014Spring_run3/BS_PCR Bio20_2014_Sprint_run3 %]ls A119_1.p1.fq@ A123_2.p1.fq@ README flowcell246_lane1_pair2_ACTTGA.fastq* A119_1.p2.fq@ A123_2.p2.fq@ flowcell246_lane1_pair1_ACTTGA.fastq* flowcell246_lane1_pair2_AGTCAA.fastq* A119_2.p1.fq@ BSmap.pl* flowcell246_lane1_pair1_AGTCAA.fastq* flowcell246_lane1_pair2_ATGTCA.fastq*

II. Run Bismark

1. run make_runBismark.sh and give the following arguments. Collect the output as a shell script to run on the queue. a. genome DIR ( /rhome/jinghuas/bigdata/bisulfide_genome/mping ) b. FQ1 ( A119_1.p2.fq ) c. FQ2 ( A119_1.p2.fq ) d. BASE ( A119_1 )

   e. is the file in which the shell script is stored ( > A119.1_runBismark.sh )

ex: one at a time: make_runBismark.sh /rhome/jinghuas/bigdata/bisulfide_genome/mping A119_1.p1.fq A119_1.p2.fq A119_1 > A119.1_runBismark.sh

ex: all at once

for i in NB_1 NB_2 A119_1 A119_2 A119_3 A123_1 A123_2 ; do make_runBismark.sh /rhome/jinghuas/bigdata/bisulfide_genome/mping ${i}.p1.fq ${i}.p2.fq ${i} output > ${i}.runBismark.sh ; done

2. submit to the queue.

ex:

qsub A119_1.runBismark.sh

III. Post process Bismark output for readability and files for Gbrowse

1. cd output
2. run extractor and process by running make_extract_convert.sh with the sample name. You will need to collect the ouput for use as a shell script. a. sample_name ( same as base ) ( A119_1 )

ex: one at a time make_extract_convert.sh A119_1 > A119_1.extract_process.sh

ex: all at once for i in NB_1 NB_2 A119_1 A119_2 A119_3 A123_1 A123_2 ; do make_extract_convert.sh $i > ${i}.extract_process.sh ; done

3. submit each to the queue

ex.

qsub A119_1.extract_process.sh

IV. Combine Data

1. make sure you are in output, same as in step III.

2. run combine script a. path of the directory with the split data ( split_methyl ) b. a file name to write the output to ( combine.txt )

   ex.

   combine_split.pl split_methyl/ > combine.txt

V. copy data to your own computer or html dir --- own computer ---

1. connect with FUGU
2. copy what you need.

---- html dir ---

1. cp onefile ~/.html/.
2. cp -r directory_name ~/.html/.