Noi/NOTES/2012-5-7: Difference between revisions

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* The libraries were sequenced on 120426_HL118 run,lane 5.
* The libraries were sequenced on 120426_HL118 run,lane 5.
* Sequencing data on genome-miner: /home/kunzhang/seqStore/120426_HL118/PE
* Sequencing data on genome-miner: /home/kunzhang/seqStore/120426_HL118/PE
== Sample index ==
== Sample indexes ==
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
Line 25: Line 25:
  less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx76.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./H1-54C
  less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx76.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./H1-54C
  less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx77.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./RC-1
  less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx77.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./RC-1
on different directory
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx7.txt.gz | /home/kunzhang/bin/trimFastq.pl 20 75 > ./PGP1F-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx45.txt.gz | /home/kunzhang/bin/trimFastq.pl  20 75 > ./PGP1iPS-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx76.txt.gz | /home/kunzhang/bin/trimFastq.pl  20 75 > ./H1-54C
== Mapping to hg19  (on Triton cluster) ==  
== Mapping to hg19  (on Triton cluster) ==  
'''Generate job files and submit jobs'''<br>
'''Generate job files and submit jobs'''<br>
Line 58: Line 63:
  qsub SEmapping_PGP1iPS-54C.job
  qsub SEmapping_PGP1iPS-54C.job
  qsub SEmapping_RC-1.job
  qsub SEmapping_RC-1.job
* I'm  supposed map the sequences to hg18. I repeated mapping again on Triton cluster
* I'm  supposed to map the sequences to hg18 and do the same thing as I did before with the previous data set to be able to compare the quality of sequencing and protocol. I repeated mapping again on Triton cluster
 
== Mapping to hg18  (on Triton cluster) ==
== Mapping to hg18  (on Triton cluster) ==
* Note: * I also trimmed 6 bp from 3' end as previously did even the quality of sequencing was good.
* Note: * I also trimmed 6 bp from 3' end as previously did even the quality of sequencing was good.
*Mapping sequences 20bp trimmed from 5'end
*Mapping sequences 20bp trimmed from 5'end
  the last two lines of SEmapping.template  
  The last two lines of SEmapping.template  
  cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20
  cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20
  ./bisMapSoapTriton.pl [FILENAME] 80
  ./bisMapSoapTriton.pl [FILENAME] 80
* generate_jobs.sh: mapping sequences 20bp trimmed from 5'end and 6bp trimmed from 3'end
* Mapping sequences 20bp trimmed from 5'end and 6bp trimmed from 3'end
  the last two lines of SEmapping.template
  The last two lines of SEmapping.template
  cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20/mapping_trimm5-20_3-6
  cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20/mapping_trimm5-20_3-6
  ../bisMapSoapTriton.pl [FILENAME] 75
  ../bisMapSoapTriton.pl [FILENAME] 75
* Then calculate specificity and on-target coverage using Dr. Zhang script
* Then calculate specificity and on-target coverage using Dr. Zhang script (on genome-miner)
'''Directories:'''
/home/nplongth/Noi_scratch/LC_120426_HL118/hg18.mapping_trim_20-75
/home/nplongth/Noi_scratch/LC_120426_HL118/hg18.mapping_trim_20-80
  /home/kunzhang/bin/bisPileupPair2coverage.pl PGP1F-54C.fwd.pileup.methylFreq.BED.txt  PGP1F-54C.fwd.pileup PGP1F-54C.rev.pileup > target_PGP1F-54C
  /home/kunzhang/bin/bisPileupPair2coverage.pl PGP1F-54C.fwd.pileup.methylFreq.BED.txt  PGP1F-54C.fwd.pileup PGP1F-54C.rev.pileup > target_PGP1F-54C
  /home/kunzhang/bin/bisPileupPair2coverage.pl PGP1iPS-54C.fwd.pileup.methylFreq.BED.txt PGP1iPS-54C.fwd.pileup PGP1iPS-54C.rev.pileup > target_PGP1iPS-54C
  /home/kunzhang/bin/bisPileupPair2coverage.pl PGP1iPS-54C.fwd.pileup.methylFreq.BED.txt PGP1iPS-54C.fwd.pileup PGP1iPS-54C.rev.pileup > target_PGP1iPS-54C
Line 90: Line 100:
|}<br>
|}<br>
'''Specificity and target coverage'''<br>
'''Specificity and target coverage'''<br>
  PGP1F-54C
  '''PGP1F-54C'''
  ###########################
  ###########################
  Total mapppable bases 676,624,800
  Total mapppable bases 676,624,800
Line 99: Line 109:
  DP>=1x 261,167 0.994596056
  DP>=1x 261,167 0.994596056
  DP>=10x 260,240 0.991065784
  DP>=10x 260,240 0.991065784
  DP>=20x 98,519 0.375187558
  DP>=20x 98,519 0.375187558
  PGP1iPS-54C
  '''PGP1iPS-54C'''
  ###########################
  ###########################
  Total mapppable bases 723,009,280
  Total mapppable bases 723,009,280
Line 109: Line 120:
  DP>=1x 209,072 0.994236366
  DP>=1x 209,072 0.994236366
  DP>=10x 208,454 0.991297483
  DP>=10x 208,454 0.991297483
  DP>=20x 100,248 0.476726712
  DP>=20x 100,248 0.476726712
 
'''H1-54C'''
  ###########################
  ###########################
  Total mapppable bases 863,980,560
  Total mapppable bases 863,980,560
Line 134: Line 147:
|}<br>
|}<br>
'''Specificity and target coverage calculation'''<br>
'''Specificity and target coverage calculation'''<br>
  PGP1F-54C
  '''PGP1F-54C'''
  ###########################
  ###########################
  Total mapppable bases 662,414,250
  Total mapppable bases 662,414,250
Line 143: Line 156:
  DP>=1x 269,065 0.9940262
  DP>=1x 269,065 0.9940262
  DP>=10x 267,989 0.990051056
  DP>=10x 267,989 0.990051056
  DP>=20x 102,254 0.377764314
  DP>=20x 102,254 0.377764314
  PGP1iPS-54C
  '''PGP1iPS-54C'''
  ###########################
  ###########################
  Total mapppable bases 700,625,100
  Total mapppable bases 700,625,100
Line 153: Line 167:
  DP>=1x 211,437 0.993548236
  DP>=1x 211,437 0.993548236
  DP>=10x 210,768 0.990404586
  DP>=10x 210,768 0.990404586
  DP>=20x 102,285 0.480640008
  DP>=20x 102,285 0.480640008
  H1-54C
  '''H1-54C'''
  ###########################
  ###########################
  Total mapppable bases 843,865,950
  Total mapppable bases 843,865,950
  Total on-target bases 724,892,066
  Total on-target bases 724,892,066
  Specificity = 0.859
  Specificity 0.859
  ###########################
  ###########################
  total capture targets 334,358 bp
  total capture targets 334,358 bp

Latest revision as of 18:06, 27 June 2012

Mapping of PGP1-F PGP1-iPS and H1 samples captured with LC Sciences probe set[edit]

Sample indexes[edit]

Sample IDs Index
PGP1-F 54C CA-2-FA.Ind7.Sol
PGP1-iPS 54C CA-2-FA.Ind45.Sol
H1 54C CA-2-FA.Ind76.Sol
RC-1 CA-2-FA.Indx77.Sol
  • Note: RC-1 is the library from Baylor
  • Refer to the previous batch (111012_HL105), the sequences were trimmed 20bp from 5' end and also 6bp from 3' end since the quality of that run was not good as usual. However the quality of this run seemed to be good. I will trim only 20bp from the 5'end.

Trimming 20 bp from 5'end (on genome-miner)[edit]

less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx7.txt | /home/kunzhang/bin/trimFastq.pl	20 80 > ./PGP1F-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx45.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./PGP1iPS-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx76.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./H1-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx77.txt | /home/kunzhang/bin/trimFastq.pl  20 80 > ./RC-1
on different directory 
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx7.txt.gz | /home/kunzhang/bin/trimFastq.pl	20 75 > ./PGP1F-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx45.txt.gz | /home/kunzhang/bin/trimFastq.pl  20 75 > ./PGP1iPS-54C
less /home/kunzhang/seqStore/120426_HL118/PE/s_5_*_Indx76.txt.gz | /home/kunzhang/bin/trimFastq.pl  20 75 > ./H1-54C

Mapping to hg19 (on Triton cluster)[edit]

Generate job files and submit jobs

  • SEmapping.template
#!/bin/csh
#PBS -q small 
#PBS -l nodes=1:ppn=2
#PBS -l walltime=12:00:00
#PBS -o [FILENAME]_SEmapping.log 
#PBS -e [FILENAME]_SEmapping.err
#PBS -V
#PBS -M nongluk6665@gmail.com 
#PBS -m abe
#PBS -A zhang-lab
cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg19
./bisReadMapperSE19Triton_b.pl [FILENAME] 80
  • generate_jobs.sh
#!/bin/bash
scriptname="qsub_script.sh"
echo "#!/bin/bash" > $scriptname
chmod +x $scriptname
for f in * 
 do
 if [ `expr index $f "."` -eq 0 ]
     then
     sed "s/\[FILENAME\]/$f/g" SEmapping.template > SEmapping_$f.job
     echo "qsub SEmapping_$f.job" >> $scriptname
 fi
done
  • qsub_script.sh
qsub SEmapping_H1-54C.job
qsub SEmapping_PGP1F-54C.job
qsub SEmapping_PGP1iPS-54C.job
qsub SEmapping_RC-1.job
  • I'm supposed to map the sequences to hg18 and do the same thing as I did before with the previous data set to be able to compare the quality of sequencing and protocol. I repeated mapping again on Triton cluster

Mapping to hg18 (on Triton cluster)[edit]

  • Note: * I also trimmed 6 bp from 3' end as previously did even the quality of sequencing was good.
  • Mapping sequences 20bp trimmed from 5'end
The last two lines of SEmapping.template 
cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20
./bisMapSoapTriton.pl [FILENAME] 80
  • Mapping sequences 20bp trimmed from 5'end and 6bp trimmed from 3'end
The last two lines of SEmapping.template
cd /home/nplongthongkum/Labdir/BSPP/Noi_Data/LC_120426_HL118.hg18_trim5-20/mapping_trimm5-20_3-6
../bisMapSoapTriton.pl [FILENAME] 75
  • Then calculate specificity and on-target coverage using Dr. Zhang script (on genome-miner)
Directories:
/home/nplongth/Noi_scratch/LC_120426_HL118/hg18.mapping_trim_20-75
/home/nplongth/Noi_scratch/LC_120426_HL118/hg18.mapping_trim_20-80

/home/kunzhang/bin/bisPileupPair2coverage.pl PGP1F-54C.fwd.pileup.methylFreq.BED.txt  PGP1F-54C.fwd.pileup PGP1F-54C.rev.pileup > target_PGP1F-54C
/home/kunzhang/bin/bisPileupPair2coverage.pl PGP1iPS-54C.fwd.pileup.methylFreq.BED.txt PGP1iPS-54C.fwd.pileup PGP1iPS-54C.rev.pileup > target_PGP1iPS-54C
/home/kunzhang/bin/bisPileupPair2coverage.pl H1-54C.fwd.pileup.methylFreq.BED.txt H1-54C.fwd.pileup H1-54C.rev.pileup > target_H1-54C

Summary of mapping data[edit]

Trimmed 20bp from 5' end[edit]

Sample IDs # of reads Total sequences (bps) # of CpGs called Read mapping rate (%)
PGP1-F 54C 22,049,038 2,204,903,800 131,416 41.86
PGP1-iPS 54C 20,159,226 2,015,922,600 105,280 48.89
H1 54C 28,060,038 2,806,003,800 162,709 41.75


Specificity and target coverage

PGP1F-54C		
###########################		
Total mapppable bases	676,624,800	
Total on-target bases	590,092,713	
Specificity	0.8721	
###########################		
total capture targets	262,586	bp
DP>=1x	261,167	0.994596056
DP>=10x	260,240	0.991065784
DP>=20x	98,519	0.375187558	
PGP1iPS-54C		
###########################		
Total mapppable bases	723,009,280	
Total on-target bases	608,068,758	
Specificity	0.841	
###########################		
total capture targets	210,284	bp
DP>=1x	209,072	0.994236366
DP>=10x	208,454	0.991297483
DP>=20x	100,248	0.476726712
H1-54C				
###########################		
Total mapppable bases	863,980,560	
Total on-target bases	745,163,756	
Specificity	0.8625	
###########################		
total capture targets	324,890	bp
DP>=1x	323,276	0.995032165
DP>=10x	322,108	0.991437102
DP>=20x	110,768	0.34094001

Trimmed 20bp from 5' end and 6bp from 3' end[edit]

Sample IDs # of reads Total sequences (bps) # of CpGs called Read mapping rate (%)
PGP1-F 54C 22,049,038 2,204,903,800 135,467 43.79
PGP1-iPS 54C 20,159,226 2,015,922,600 106,535 50.69
H1 54C 28,060,038 2,806,003,800 167,445 43.59


Specificity and target coverage calculation

PGP1F-54C		
###########################		
Total mapppable bases	662,414,250	
Total on-target bases	576,137,096	
Specificity	0.8698	
###########################		
total capture targets	270,682	bp
DP>=1x	269,065	0.9940262
DP>=10x	267,989	0.990051056
DP>=20x	102,254	0.377764314
PGP1iPS-54C		
###########################		
Total mapppable bases	700,625,100	
Total on-target bases	583,970,343	
Specificity	0.8335	
###########################		
total capture targets	212,810	bp
DP>=1x	211,437	0.993548236
DP>=10x	210,768	0.990404586
DP>=20x	102,285	0.480640008	
H1-54C		
###########################		
Total mapppable bases	843,865,950	
Total on-target bases	724,892,066	
Specificity	0.859
###########################		
total capture targets	334,358	bp
DP>=1x	332,505	0.994458036
DP>=10x	331,175	0.990480264
DP>=20x	114,674	0.342967717
  • Note: actually there is no need to map RC-1 sample since this library was not capture with bisulfite padlock probe.