Noi/NOTES/2014-5-24: Difference between revisions

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==== Directional option ====
==== Directional option ====
* In folder '''NP.RRBS.Trim1_directional'''
* In folder '''NP.RRBS.Trim1_directional'''
* I am not authorized to install pip, so there were some difficulties to install functional cutadapt. My friend helped me to generate virtual environment, and it needs to be activated when running cutadapt and trim_galore.
* I am not authorized to install python-pip, so there were some difficulties to install functional cutadapt since the library was missing. My friend helped me to generate virtual environment, and it needs to be activated when running cutadapt and trim_galore.
** Activate environment: source ~/env/bin/activate
** Activate environment: source ~/env/bin/activate
** Deactivate environment: deactivate
** Deactivate environment: deactivate
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| 10nu_apr10_1to50_no2||4,601,068||3,012,729||65.48
| 10nu_apr10_1to50_no2||4,601,068||3,012,729||65.48
|}
|}
==== Non-directional option ====
==== Non-directional option ====
* I also ran trim_galore with ''--non_directional'' option.  "This option will screen quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read and, if found, removes the first two basepairs. Like with the option '--rrbs' this avoids using cytosine positions that were filled-in during the end-repair step. '--non_directional' requires '--rrbs' to be specified as well" (Krueger F, 2011).
* I also ran trim_galore with ''--non_directional'' option.  "This option will screen quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read and, if found, removes the first two basepairs. Like with the option '--rrbs' this avoids using cytosine positions that were filled-in during the end-repair step. '--non_directional' requires '--rrbs' to be specified as well" (Krueger F, 2011).

Revision as of 19:10, 25 May 2014

RRBS data analysis

  • Data information
    • scRRBS and 10nuclei RRBS data generated on 2014-04-25 [[1]]
    • Working directory on genome-miner: /home/nplongth/Noi_scratch/2014_works/RRBS_data/RRBS_140514_HL166

Step 1: Adapter trimming and quality filtering

Tool: Trim Galore from Babrahan Bioinformatic which is a wrapper script that use Cutadapt for adapter trimming and fastQC for quality filtering

  • By Running checkphred.pl, I confirm that all sequencing data are phred64

Directional option

  • In folder NP.RRBS.Trim1_directional
  • I am not authorized to install python-pip, so there were some difficulties to install functional cutadapt since the library was missing. My friend helped me to generate virtual environment, and it needs to be activated when running cutadapt and trim_galore.
    • Activate environment: source ~/env/bin/activate
    • Deactivate environment: deactivate
#!/bin/bash

for f in ../RRBS_140514_HL166.rawdata/*.txt
 
do 

 ../trim_galore -q 20 --phred64 -a AGATCGGAAGAGC --rrbs $f

done
- Quality Phred score cutoff: 20
- Quality encoding type selected: ASCII+64
- Adapter sequence: 'AGATCGGAAGAGC'
- Maximum trimming error rate: 0.1 (default)
- Minimum required adapter overlap (stringency): 1 bp (most stringency!!)
- Minimum required sequence length before a sequence gets removed: 20 bp
- File was specified to be an MspI-digested RRBS sample. Sequences with adapter contamination will be trimmed a further 2 bp to remove potential methylation-biased bases from the end-repair reaction
Samples # of reads # of read after trimming % Remaining reads
apr10_1to50_no1 5,659,042 3,996,745 70.63
apr10_1to50_no2 5,707,396 3,863,720 67.70
apr10_1to50_no3 5,492,909 3,922,931 71.42
apr10_1to50_no4 5,351,954 3,526,832 65.90
apr10_1to50_no5 3,605,391 2,454,517 68.08
apr10_1to50_no6 4,027,892 2,651,359 65.82
apr10_1to100_no7 3,352,177 2,250,422 67.13
apr10_1to100_no8 2,071,082 1,428,535 68.98
apr10_1to100_no9 2,752,431 1,929,729 70.11
apr10_1to100_no10 2,203,299 1,424,776 64.67
10nu_apr10_1to50_no1 4,527,606 3,163,200 69.86
10nu_apr10_1to50_no2 4,601,068 3,012,729 65.48

Non-directional option

  • I also ran trim_galore with --non_directional option. "This option will screen quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read and, if found, removes the first two basepairs. Like with the option '--rrbs' this avoids using cytosine positions that were filled-in during the end-repair step. '--non_directional' requires '--rrbs' to be specified as well" (Krueger F, 2011).
  • In folder NP.RRBS.Trim2_non-directional
#!/bin/bash

for f in ../RRBS_140514_HL166.rawdata/*.txt

 do 
	../trim_galore -q 20 --phred64 -a AGATCGGAAGAGC --rrbs --non_directional $f

done

Step 2: Mapping

Tool: Bismark

Step 2.1: Running bismark_genome_preparation

  • In directory: /home/nplongth/hg19_Bismark/, I copied hg reference genome sequences from /home/kunzhang/HsGenome/hg19/HsGenome19.fa
../softwares/bismark_v0.12.2/bismark_genome_preparation --path_to_bowtie /home/kunzhang/softwares/bowtie-0.12.8/ --verbose /home/nplongth/hg19_Bismark/
  • It generated Bisulfite_Genome folder, including two sub-folders CT_conversion and GA_conversion

Directional mapping

  • In folder NP.RRBS.Trim1_directional/Bismark-run.directional
Run.bismark.directional.sh

#!/bin/bash

for f in ../*.fq

do 

	/home/nplongth/softwares/bismark_v0.12.2/bismark --path_to_bowtie /home/nplongth/softwares/bowtie-0.12.8 -n 1 /home/nplongth/hg19_Bismark/ $f

done