Noi/NOTES/2012-4-27

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  • Link to calendar: [[1]]

Check chromosome and coordinate of CpG and SNP derived from mapping by bisReadMapperSE19Triton and from Robert's ASM pipeline[edit]

  • bisReadMapperSE19Triton mapping
  • 509557 GK0005-001.hg19.BED.txt
sort GK0005-001.hg19.BED.txt > sorted_chrpos_GK0005-001.hg19.BED.txt
509557 sorted_chrpos_GK0005-001.hg19.BED.txt
track name="GK0005-001_hg19" description="Methylation level" visibility=2 useScore=1 itemRgb="On"
chr10	100017297	100017298	0.983	58	+	100017297	100017298	210,0,0
chr10	100017304	100017305	0.930	57	+	100017304	100017305	210,0,0
chr10	100017325	100017326	1.000	58	+	100017325	100017326	210,0,0
chr10	100017333	100017334	0.964	55	+	100017333	100017334	210,0,0
chr10	100029179	100029180	0.951	41	+	100029179	100029180	210,0,0
chr10	100029247	100029248	0.450	40	+	100029247	100029248	90,150,0
chr10	100029342	100029343	0.026	39	+	100029342	100029343	0,240,0
chr10	100029476	100029477	0.897	58	+	100029476	100029477	210,0,0
chr10	100029482	100029483	0.276	58	+	100029482	100029483	60,180,0
  • Robert's pipeline
488563 CpGMethFreq.GK0005001.bed
sort CpGMethFreq.GK0005001.bed > sorted_CpGMethFreq.GK0005001.bed
488563 sorted_CpGMethFreq.GK0005001.bed
track name="GK0005001 CpG MF" description="GK0005001 CpG Methylation Frequency" visibility="dense" useScore=1 itemRgb="On"
chr10	100017297	100017298	0.982	57	+	100017297	100017298	210,0,0
chr10	100017304	100017305	0.929	56	+	100017304	100017305	210,0,0
chr10	100017325	100017326	0.999	57	+	100017325	100017326	210,0,0
chr10	100017333	100017334	0.965	57	+	100017333	100017334	210,0,0
chr10	100029179	100029180	0.950	40	+	100029179	100029180	210,0,0
chr10	100029247	100029248	0.450	40	+	100029247	100029248	90,150,0
chr10	100029342	100029343	0.026	39	+	100029342	100029343	0,240,0
chr10	100029476	100029477	0.897	58	+	100029476	100029477	210,0,0
chr10	100029482	100029483	0.276	58	+	100029482	100029483	60,180,0
  • The number of called CpG by Robert's pipeline was lower than bisReadMapperSE19Triton. This is possible that Robert's pipeline is more stringent than bisReadMapperSE19Triton, so the number of called CpG was lower. However the chromosome positions, CpG coordinates and methylation frequency were consistent.

Check the CpG coordination in ASM output file[edit]

  • To check if the CpG coordinate from ASM analysis is on start or end position.
    • CpG coordinates in methylFreqMatrix generated by the BED file mapped by bisReadMapperSE19Triton are on the end position
awk '{print $1"\t"$2}' UCLA-4batches_methyl-noSNP_min76_minSTD0.1.txt | head -3
0chr_position	GK0005-001
chr13:45885186	0.701
chr7:105318897	0.680
less GK0005-001.hg19.BED.txt
chr13   45885185        45885186        0.701   144     +       45885185        45885186        150,90,0
chr7    105318896       105318897       0.680   25      +       105318896       105318897       150,90,0
  • File's description:
(9) MFASMAnalysisPValues.$expname.$asmtesttype.txt 
1st field is the SNP coordinate,
2nd field is the SNP call, 
3rd field is the strandedness of the SNP call (ss - single strand ds - double strand), 
4th field is the strand of the SNP call,
5th field is the type of calculation (a number stands for the coordinate of a specific CpG. Stable and stable average are defined above). 
6th field shows the difference in methylation frequency values between CpGs on SNP bases 1 and 2
7th field is the p-value associated with the observed allele specific differential methylation values. 
8th and 9th are the number of C's and T's associated with SNP base1, respectively. 
Fields 10 and 11 are the number of C's and T's associated with SNP base2, respectively.
MFASMAnalysisPValues.GK0005001.cpg.txt 
3:173374	AG	ss	-	173345	-0.0262626262626262	0.783557175124665	36	8	38	7
CpGMethFreq.GK0005001.bed
chr3    173344  173345  0.831   89      +       173344  173345  180,60,0
MFASMAnalysisPValues.GK0005001.cpg.txt
20:62405863	CG	ss	-	62405854	 0.153846153846154	0.402328024619046	22	4	9	4
CpGMethFreq.GK0005001.bed
chr20   62405853        62405854        0.810   42      +       62405853        62405854        180,60,0
  • This means that the CpG coordinates from ASM analysis are on the end position as the CpG in methylFreqMatrix as well

Check SNP[edit]

  • In the SNP file from bisReadMapperSE19Triton mapping
(1) chromosome name
(2) position
(3) reference base call
(4) strand (Watson/Crick)
(5) variant call
(6) variant quality
(7) total read depth
(8,10,12..etc) depth of sequenced base
(9,11,13..etc) base sequenced
  • SNPs before filtered
chr12   25537917        A       C       A       157     44      A       44
chr16   84539303        C       C       K       49      21      T       6       G       15
chr2    44588941        G       W       R       94      31      A       16      G       15
  • SNP after filtered
19,781 GK0005-001.fwd.pileup.snp.filtered
head 3 GK0005-001.fwd.pileup.snp.filtered
SNP position	SNP call	SNP qual	dbSNP	RefAlleles	SNP call(fwd)	Allele count(fwd)	SNP call(rev)	Allele count(rev)
chr12:25537917	T	157	rs923491	K	-	-	A	A,44	
chr16:84539303	S	49	rs12716753	S	-	-	K	T,6,G,15
chr2:44588941	R	94	rs698775	R	R	A,16,G,15	-	-
  • SNP called by Robert's pipeline
18,146 SNPs.GK0005001.txt
chr12   25537917        A       AA      homozygous      -       ss
chr16   84539303        CG      TG      heterozygous    -       ss
chr2    44588941        AG      AG      heterozygous    +       ss
  • SNPDB
Assembly Genome_Build Chr Chr_Pos
rs923491   --> GRCh37.p5	37.3	12	25537917 RefSNP Alleles:	G/T (check)
rs12716753 --> GRCh37.p5	37.3	16	84539303 RefSNP Alleles:	C/G
rs698775   --> GRCh37.p5	37.3	2	44588941 RefSNP Alleles:	A/G
  • The results above revealed that the SNP called by both methods were consistent