RRBS甲基化分析流程

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RRBS甲基化流程

分析流程

和普通的测序分析一致,首先fastqc质量检测,接着对序列进行修剪,修剪后再质量检测;如果质量检测通过,则进行序列回帖,然后去除重复,计算甲基化程度,以及一些后续分析,本次后续分析使用R包methlykit以及edmr,还有其他一些甲基化分析软件可以参见附录。
在这里插入图片描述

流程命令

比对流程

首先进行fastqc质量鉴定,如果需要的话,再去除接头,PS:去除接头后需要重新进行质量鉴定
接下来就是Bismark序列比对,Bismark会通过调用bowtie1或者bowtie2进行比对,(生信菜鸟团有人介绍使用bsmap进行序列比对)
下面以调用bowtie1为例,

1、准备基因组

bismark_genome_preparation --bowtie1 /home/huangml/WangYunpeng/RRBS/

--bowtie1 选定bowtie1,如果bowtie不在环境变量路径内,还要另外指明

/home/huangml/WangYunpeng/RRBS/ 选定参考基因组位置

2、测序文件比对

bismark -bowtie1 /home/huangml/WangYunpeng/RRBS -1 P11m414_LiNew_R1.fq.gz -2 P11m414_LiNew_R2.fq.gz

-1和-2 分别选定双端测序文件

/home/huangml/WangYunpeng/RRBS/ 选定参考基因组位置

3、去除重复序列(消除PCR过度扩增的影响)

deduplicate_bismark --bam P3m76_LiNew_R1_bismark_pe.bam

--bam 保证输出为bam格式

4、抽提出甲基化的统计信息

bismark_methylation_extractor --bedGraph --gzip P3m76_LiNew_R1_bismark_pe.deduplicated.bam

--gzip 结果输出为GZIP压缩文件

--bedGraph 结果输出格式为bedGraph

5、甲基化的可视化 (此步骤需要用Bismark在GitHub上的版本,官网版本存在bug,图片会无法显示)

bismark2report

需要用root权限在上一步统计信息的文件夹下面运行,生成HTML格式的图形统计结果

详细版的使用Bismark使用参数在https://rawgit.com/FelixKrueger/Bismark/master/Docs/Bismark_User_Guide.html
该软件的作者还有相关练习在http://www.bioinformatics.bbsrc.ac.uk/training.html
后续分析:
如果是使用methlykit包进行分析,可以将Bismark比对生成的bam文件转成sam,并且排序(此处的bam文件最好是不要经过deduplicate_bismark 去除重复序列的。如果去除重复序列,methlykit聚类图效果不好,可以两个都试一试),methlykit包中有提供processBismarkAln函数,对其进行预处理,即可使用methlykit包进行分析
事实上,bismark不仅仅针对RRBS测序,还可以供其他建库方式比对使用。
下面是用不同建库方式的时候,使用Bismark时候的建议
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可视化:

Bismark作者提供了SeqMonk工具进行可视化,同样的,有相关练习http://www.bioinformatics.bbsrc.ac.uk/training.html

差异甲基化的分析方法

methylKit包,本次使用的就是这个,滑动窗口选取差异甲基化区域
eDMR包(可以在GitHub上搜索,与methlykit作者相同)比起methylKit滑动窗口选取差异甲基化区域,该方法用“双峰正态模型”将邻近的一些CpG划分为一片甲基化区域
PS:如果是多组样本做overlap取甲基化区域的交集,eDMR分析结果不同组样本比较的时候,区域起始与结束位置并不一致,overlap难以选取,methylKit结果可以稍微容易一点选取overlap,但是做实验的时候,更希望选出差异甲基化的promoter区域,因此多轮筛选出来结果也不是很好。如果要针对promoter区域分析差异甲基化,可以使用GRanges对象,自己编写程序统计,附录有demo版本的代码。
还有一些其他软件包如下:
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methylKit 相关统计分析

首先,将Bismark比对结果的sam文件,先使用samtools进行排序,然后用processBismarkAln函数读入R,预处理生成类似如下的文件:

  • P11m414_CpG.txt

  • P11m415_CpG.txt

  • P11m416_CpG.txt

    library(methylKit)
    setwd("/home/huangml/WangYunpeng/RRBS/result")
    mySaveFolder="/home/huangml/WangYunpeng/RRBS/result/2-methylKit-result"
    由sam文件创建可以读入的文件
    my.methRaw=processBismarkAln(location = “P11m414_LiNew/P11m414_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P11m414”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)
    my.methRaw=processBismarkAln(location = “P11m415_LiNew/P11m415_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P11m415”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)
    my.methRaw=processBismarkAln(location = “P11m416_LiNew/P11m416_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P11m416”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)
    my.methRaw=processBismarkAln(location = “P3m76_LiNew/P3m76_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P3m76”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)
    my.methRaw=processBismarkAln(location = “P3m77_LiNew/P3m77_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P3m77”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)
    my.methRaw=processBismarkAln(location = “P3m78_LiNew/P3m78_LiNew_R1_bismark_pe.sorted.sam”,
    sample.id=“P3m78”, assembly=“M.musculus”,
    read.context=“CpG”,save.folder=mySaveFolder)

文件具体内容如下:

chrBase	chr	base	strand	coverage	freqC	freqT
chr1.3020813	chr1	3020813	F	10	100.00	0.00
chr1.3020841	chr1	3020841	F	19	0.00	100.00
chr1.3020876	chr1	3020876	F	47	82.98	17.02
chr1.3020890	chr1	3020890	F	38	97.37	2.63
chr1.3020944	chr1	3020944	F	49	34.69	65.31
chr1.3020970	chr1	3020970	F	22	63.64	36.36
chr1.3020986	chr1	3020986	F	22	81.82	18.18
chr1.3021011	chr1	3021011	F	13	30.77	69.23
chr1.3020877	chr1	3020877	R	196	17.86	82.14

因为bowtie选取的基因组,除了Chr1,Chr2这些染色体外,还有JH584304.1这种染色体,为了后续分析的方便,使用shell脚本将这些行去除

cat P11m414_CpG.txt|grep chr >P11m414_CpG_filter.txt
cat P11m415_CpG.txt|grep chr >P11m415_CpG_filter.txt
cat P11m416_CpG.txt|grep chr >P11m416_CpG_filter.txt
cat P3m76_CpG.txt|grep chr >P3m76_CpG_filter.txt
cat P3m77_CpG.txt|grep chr >P3m77_CpG_filter.txt
cat P3m78_CpG.txt|grep chr >P3m78_CpG_filter.txt

此处strand列:
R在后面的格式中为"-",表示CpG在负链上;F在后面的格式中为"+",表示CpG在正链上
freqC、freqT为C、T出现的频率,coverage列指的是:覆盖度
例如:coverage为12,freqC为25,freqT为75,即所有的reads中,有12X25%=3个在此处为C;有12X75%=9个在此处为T。
因为亚硫酸氢钠处理后, 正常的C会转成T,甲基化的C不改变,
所以,在这里,每个位点甲基化的概率就可以求出来,就是这里的freqC
注意:输入文件此处base的位置,是从0开始,C的坐标

预处理完成后就可以愉快地进行分析了
在这里插入图片描述
在这幅图中,横坐标表示甲基化的概率,纵坐标表示此概率中位点个数
甲基化的概率是由每个位点上,测到的C的数量÷此位点测到的(C+T)数量(即coverage覆盖度 ),就是freqC
比如有23.5%的位点是0-10%概率被甲基化
由于一个位点,要么完全甲基化,要么完全没有甲基化,所以这个图应该呈“两头高,中间低 ”
然后就是每个CpG覆盖度的图,横坐标表示覆盖度,纵坐标表示该覆盖度的CpG的比例。按理来说,应该是随着覆盖度的增加,CpG占得比例越低,也就是如下图,只有一个峰
在这里插入图片描述
我这边实际做的时候有的样本在右侧出现了第二个峰,即疑似收到PCR扩增影响
在这里插入图片描述

还有样本相关度图
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样本聚类图
在这里插入图片描述
PCA碎石图(反应PCA各个主成分占有的信息量)
[外链图片转存失败,源站可能有防盗链机制,建议将图片保存下来直接上传(img-V26ZXkDc-1586003835884)(https://i.imgur.com/j924Zyp.png)]

PCA图
在这里插入图片描述
接下来就是差异甲基化分析,以及一些注释,
注释文件可以在UCSC Table Browser上面下载
http://genome.ucsc.edu/cgi-bin/hgTables
在这里插入图片描述
差异甲基化分析也就是调用一个函数就可以完成,然后也能绘制高低甲基化的CpG在各个染色体上面的分布
在这里插入图片描述
也能做出来差异甲基化位点所处区域的注释饼图
在这里插入图片描述
在这里插入图片描述
计算差异甲基化区域的时候,用getMethylDiff选取阀值,为了后续方便,difference选0,qvalue选1,把所有的差异甲基化信息都取出来,方便后续做overlap

# get hyper methylated bases
myDiff25p.hyper=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hyper")
## get hypo methylated bases
myDiff25p.hypo=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hypo")
### get all differentially methylated bases
myDiff25p=getMethylDiff(myDiff,difference=25,qvalue=0.01)

最后可以做出来一张表
dist.to.feature表示距离对应转录本的距离,此处的甲基化程度是计算了加权平均,即:
[(样本1的coverage X 样本1的甲基化程度) + (样本2的coverage X 样本2的甲基化程度) + (样本3的coverage X 样本3的甲基化程度)] / 全部样本的coverage
而且p值计算也是跟coverage有关,一般coverage越高,p值越小,因此最后筛选的时候,需要选取一个甲基化差异大小的cutoff值才能筛出较好的结果。

target.row	dist.to.feature	feature.name	feature.strand	prom	exon	intron	CpGi	shores	chr	start	end	strand	pvalue	qvalue	meth.diff	chr.1	start.1	end.1	strand.1	coverage1	numCs1	numTs1	coverage2	numCs2	numTs2	coverage3	numCs3	numTs3	coverage4	numCs4	numTs4	coverage5	numCs5	numTs5
1	-35253	ENSMUST00000193812.1	+	0	0	0	0	0	chr1	3037501	3038000	*	0.130978182587743	0.368847571732731	2.65453921631286	chr1	3037501	3038000	*	589	450	139	766	584	182	467	331	136	11	9	2	619	468	151
2	-27753	ENSMUST00000193812.1	+	0	0	0	0	0	chr1	3045001	3045500	*	0.205092438212255	0.464935651021862	6.46069192334451	chr1	3045001	3045500	*	18	16	2	79	59	20	108	75	33	160	111	49	58	45	13
3	19487	ENSMUST00000082908.1	+	0	0	0	0	0	chr1	3121501	3122000	*	0.473963234904003	0.671055466685657	1.75762409327408	chr1	3121501	3122000	*	405	135	270	381	124	257	399	123	276	28	11	17	243	75	168
4	42487	ENSMUST00000082908.1	+	0	0	0	0	0	chr1	3144501	3145000	*	0.000500421870756168	0.00770695458755044	34.2622950819672	chr1	3144501	3145000	*	20	17	3	10	10	0	21	19	2	30	7	23	10	8	2
5	47987	ENSMUST00000082908.1	+	0	0	0	0	0	chr1	3150001	3150500	*	0.378680177157077	0.614693649805443	3.04931554931556	chr1	3150001	3150500	*	97	69	28	199	138	61	139	90	49	132	90	42	191	129	62
6	23550	ENSMUST00000195335.1	-	0	0	1	0	0	chr1	3344501	3345000	*	0.384323438187859	0.618361247561513	0.888609129944173	chr1	3344501	3345000	*	1546	1188	358	1969	1505	464	1558	1185	373	423	323	100	1463	1100	363
7	-16995	ENSMUST00000192973.1	-	0	0	1	1	1	chr1	3531501	3532000	*	0.321950144928481	0.573991640642017	4.68823747978991	chr1	3531501	3532000	*	104	47	57	10	2	8	542	152	390	630	240	390	575	277	298
8	499	ENSMUST00000070533.4	-	1	1	1	1	1	chr1	3670501	3671000	*	0.0400319523622198	0.181718957464176	1.20385138561919	chr1	3670501	3671000	*	564	21	543	875	37	838	774	27	747	793	17	776	1157	33	1124
9	0	ENSMUST00000070533.4	-	1	1	0	1	1	chr1	3671001	3671500	*	0.235280154140887	0.496408529219308	0.329122071544707	chr1	3671001	3671500	*	1440	34	1406	2316	25	2291	911	6	905	1347	14	1333	1205	23	1182
10	10848	ENSMUST00000193244.1	+	0	0	0	0	0	chr1	3691001	3691500	*	0.0627064903720938	0.24089809047917	12.4561403508772	chr1	3691001	3691500	*	25	4	21	35	14	21	41	7	34	41	1	40	32	12	20
11	-11010	ENSMUST00000194454.1	+	0	0	0	0	0	chr1	3740501	3741000	*	0.554821487335487	0.70972185690377	2.88107656528709	chr1	3740501	3741000	*	67	44	23	104	56	48	75	41	34	79	46	33	105	57	48
12	13993	ENSMUST00000194454.1	+	0	0	0	0	0	chr1	3766001	3766500	*	0.314915932386795	0.56842668204026	5.08922670191673	chr1	3766001	3766500	*	55	31	24	98	53	45	71	35	36	119	59	60	77	39	38
13	-35069	ENSMUST00000157708.2	-	0	0	0	0	0	chr1	3819001	3819500	*	0.492413307552576	0.680133325397598	1.23209686005095	chr1	3819001	3819500	*	806	486	320	675	404	271	874	513	361	31	15	16	607	362	245

edmr 相关统计分析####

edmr是同一作者开发的分析软件包,需要导入methylKit的结果对象,结果也能做出来一张表

X.seqnames	X.start	X.end	X.width	X.strand	X.mean.meth.diff	X.num.CpGs	X.num.DMCs	X.DMR.pvalue	X.DMR.qvalue	mean.meth.diff	num.CpGs	num.DMCs	DMR.pvalue	DMR.qvalue	gene_id
chr1	3144713	3144714	2	*	34.2622950819672	1	1	0.0271046807426123	0.0317195618915124	34.2622950819672	1	1	0.0271046807426123	0.0317195618915124	ENSMUSG00000064842
chr1	4440613	4440614	2	*	35.1841820151679	1	1	0.00632050671943642	0.00994437732842689	35.1841820151679	1	1	0.00632050671943642	0.00994437732842689	ENSMUSG00000025902
chr1	4708929	4708930	2	*	42.707672796449	1	1	0.000274707245588477	0.000838466762644724	42.707672796449	1	1	0.000274707245588477	0.000838466762644724	ENSMUSG00000088000
chr1	7335729	7335730	2	*	27.027027027027	1	1	0.0110216692220077	0.0155357692771628	27.027027027027	1	1	0.0110216692220077	0.0155357692771628	ENSMUSG00000097797
chr1	7961488	7961489	2	*	42.5840474620962	1	1	0.000921281050334225	0.00222516022281138	42.5840474620962	1	1	0.000921281050334225	0.00222516022281138	ENSMUSG00000025909
chr1	11266003	11266118	116	*	13.3487227902122	4	1	0.0997579489181488	0.100014616025142	13.3487227902122	4	1	0.0997579489181488	0.100014616025142	ENSMUSG00000048960
chr1	14034006	14034091	86	*	8.68367639534687	2	1	0.0105262967680302	0.0149942009607968	8.68367639534687	2	1	0.0105262967680302	0.0149942009607968	ENSMUSG00000089358
chr1	14482427	14482428	2	*	-38.6322188449848	1	1	0.000588236522659868	0.00153378846466034	-38.6322188449848	1	1	0.000588236522659868	0.00153378846466034	ENSMUSG00000025932
chr1	16881507	16881509	3	*	-15.5697317965766	2	1	0.000998453592196275	0.00236273734671548	-15.5697317965766	2	1	0.000998453592196275	0.00236273734671548	ENSMUSG00000091020
chr1	17947597	17947599	3	*	-8.78092667566352	2	1	0.0219766732675353	0.0268544227000858	-8.78092667566352	2	1	0.0219766732675353	0.0268544227000858	ENSMUSG00000025774
chr1	19283718	19283719	2	*	45.9718969555035	1	1	5.65448522173639e-05	0.000230050227825624	45.9718969555035	1	1	5.65448522173639e-05	0.000230050227825624	ENSMUSG00000025927
chr1	22078450	22078451	2	*	-26.024011299435	1	1	0.0212495775275785	0.0261849151051362	-26.024011299435	1	1	0.0212495775275785	0.0261849151051362	ENSMUSG00000097109
chr1	25933225	25933273	49	*	7.14092324883044	3	1	0.00102059992978815	0.00239574561831797	7.14092324883044	3	1	0.00102059992978815	0.00239574561831797	ENSMUSG00000052558
chr1	33305755	33305756	2	*	31.25	1	1	0.00926663084236262	0.0134958780582083	31.25	1	1	0.00926663084236262	0.0134958780582083	ENSMUSG00000065223

做完注释以后的表

gene_id	X.seqnames	X.start	X.end	X.width	X.strand	mean.meth.diff	num.CpGs	num.DMCs	DMR.pvalue	DMR.qvalue	external_gene_name	description
ENSMUSG00000000078	chr13	5904326	5904437	112	*	22.3150215456186	2	1	0.00525297729046551	0.00859482460523443	Klf6	Kruppel-like factor 6 [Source:MGI Symbol;Acc:MGI:1346318]
ENSMUSG00000000103	chrY	1825395	1825676	282	*	-18.9428561030264	2	1	0.0582084675097316	0.0603777271684794	Zfy2	zinc finger protein 2, Y-linked [Source:MGI Symbol;Acc:MGI:99213]
ENSMUSG00000000142	chr11	108934666	108934880	215	*	-10.9727382510561	3	1	2.78572193216407e-05	0.000132378412142268	Axin2	axin 2 [Source:MGI Symbol;Acc:MGI:1270862]
ENSMUSG00000000168	chr9	50646206	50646207	2	*	-32.1727549467276	1	1	0.000364708428440863	0.00104527776398199	Dlat	dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) [Source:MGI Symbol;Acc:MGI:2385311]
ENSMUSG00000000184	chr6	127123258	127123259	2	*	-25.9238677410392	1	1	0.0498456787199272	0.0522285017241664	Ccnd2	cyclin D2 [Source:MGI Symbol;Acc:MGI:88314]
ENSMUSG00000000214	chr7	142896455	142896594	140	*	19.7056100872936	4	1	0.00416596289719983	0.00717937684544462	Th	tyrosine hydroxylase [Source:MGI Symbol;Acc:MGI:98735]
ENSMUSG00000000282	chr11	74842332	74843170	839	*	-5.32093188731543	12	3	2.05083914787464e-06	1.44423552040088e-05	Mnt	max binding protein [Source:MGI Symbol;Acc:MGI:109150]
ENSMUSG00000000305	chr2	179539423	179539682	260	*	11.883850699595	3	1	0.00253251875295475	0.00488793676306709	Cdh4	cadherin 4 [Source:MGI Symbol;Acc:MGI:99218]
ENSMUSG00000000305	chr2	179735469	179735470	2	*	25.2322880371661	1	1	0.033753452531274	0.0379645150824816	Cdh4	cadherin 4 [Source:MGI Symbol;Acc:MGI:99218]
ENSMUSG00000000320	chr11	70277150	70277152	3	*	-6.357836313962	2	1	0.00184192025034553	0.00379085347298226	Alox12	arachidonate 12-lipoxygenase [Source:MGI Symbol;Acc:MGI:87998]
ENSMUSG00000000532	chr15	101204648	101204783	136	*	12.0686133893593	5	1	0.0404981742974062	0.0439984811837062	Acvr1b	activin A receptor, type 1B [Source:MGI Symbol;Acc:MGI:1338944]
ENSMUSG00000000617	chr11	50859302	50859426	125	*	-15.4346198344394	4	1	0.00203672422170811	0.00408860437637684	Grm6	glutamate receptor, metabotropic 6 [Source:MGI Symbol;Acc:MGI:1351343]
ENSMUSG00000000628	chr6	82824502	82824503	2	*	35.8208955223881	1	1	0.00426469215430179	0.00731716016885342	Hk2	hexokinase 2 [Source:MGI Symbol;Acc:MGI:1315197]
ENSMUSG00000000631	chr11	77844743	77844744	2	*	41.6293810589113	1	1	1.44892991507138e-06	1.06752042272171e-05	Myo18a	myosin XVIIIA [Source:MGI Symbol;Acc:MGI:2667185]
ENSMUSG00000000631	chr11	77850325	77850371	47	*	8.51951370771973	2	1	0.062903399303469	0.0646358691348567	Myo18a	myosin XVIIIA [Source:MGI Symbol;Acc:MGI:2667185]
ENSMUSG00000000673	chr17	83846617	83846720	104	*	13.7457563735597	5	1	0.000305531979488168	0.000914249703127139	Haao	3-hydroxyanthranilate 3,4-dioxygenase [Source:MGI Symbol;Acc:MGI:1349444]
ENSMUSG00000000794	chr3	89678068	89678150	83	*	7.53771410606525	4	1	0.00138514401187844	0.00300786380783757	Kcnn3	potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 [Source:MGI Symbol;Acc:MGI:2153183]
ENSMUSG00000000794	chr3	89539229	89539352	124	*	9.2559368065671	3	1	0.00160395436916688	0.00337436591041887	Kcnn3	potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 [Source:MGI Symbol;Acc:MGI:2153183]
ENSMUSG00000000805	chr11	84959957	84960110	154	*	11.5381727783586	7	1	0.00162022659053642	0.00340247584012648	Car4	carbonic anhydrase 4 [Source:MGI Symbol;Acc:MGI:1096574]
ENSMUSG00000000823	chr2	181589875	181589993	119	*	-14.4753926701571	2	1	3.06269548409202e-05	0.000142075040512046	Zfp512b	zinc finger protein 512B [Source:MGI Symbol;Acc:MGI:2685478]

主要内容就是那几个txt表,生成的都是统计模型相关的图片
表各列说明:seqnames,start,end,width,strand指定差异甲基化区域的位置
mean.meth.diff甲基化差异程度
num.CpGs该区域内CpG的个数
num.DMCs该区域内CpG中差异甲基化的个数
DMR.pvalue和DMR.qvalue是差异甲基化的p值和q值
筛选条件DMC.qvalue = 0.05,num.DMCs = 1,num.CpGs = 1,DMR.methdiff = 5
注释最近基因名是根据"转录起始位点",所以会有在A基因里,注释名称为B的情况
文献链接:http://europepmc.org/articles/PMC3622633

Promoter甲基化程度分析####

对于实验人员来说,其实上述两张表在进行多次overlap之后,再选取P值cut off后,已经只有很少一部分,然而这些交集部分,又往往会出现在exon区,intron区,甚至距离基因十万八千里的基因间区域,这就要求我们自行编写程序对promoter区域的CpG甲基化程度进行分析。
首先,取出methlykit预处理过后生成的,每个样本CpG甲基化程度文件。(就是那个Bismark比对sam文件,使用samtools进行sort之后,使用methlykit包中函数processBismarkAln生成的txt文件,注意,最好先用grep去除JH584304.1这种染色体,方便后续的分析)

chrBase	chr	base	strand	coverage	freqC	freqT
chr1.3020841	chr1	3020841	F	15	0.00	100.00
chr1.3020876	chr1	3020876	F	55	83.64	16.36
chr1.3020890	chr1	3020890	F	48	87.50	12.50
chr1.3020944	chr1	3020944	F	51	15.69	84.31
chr1.3020970	chr1	3020970	F	10	70.00	30.00
chr1.3020986	chr1	3020986	F	10	80.00	20.00
chr1.3020877	chr1	3020877	R	174	15.52	84.48
chr1.3020891	chr1	3020891	R	167	85.63	14.37
chr1.3020945	chr1	3020945	R	221	56.56	43.44

写了一些代码进行了初步的统计,代码参考附录,取了每个转录本转录起始位点上游200到下游1500bp范围内的CpG,根据Coverage计算出加权平均值,结果文件如下,value值为该转录本promoter区甲基化程度。如果为NA,则该promoter区没有测出CpG。

seqnames	start	end	width	strand	value	transUp$tx_id	transUp$tx_name
chr1	3052733	3054433	1701	+	NA	1	ENSMUST00000160944
chr1	3100516	3102216	1701	+	NA	2	ENSMUST00000082908
chr1	3465087	3466787	1701	+	NA	3	ENSMUST00000161581
chr1	4527517	4529217	1701	+	NA	4	ENSMUST00000180019
chr1	4806288	4807988	1701	+	1.95143414634146	5	ENSMUST00000134384
chr1	4806323	4808023	1701	+	1.91739263803681	6	ENSMUST00000027036
chr1	4806392	4808092	1701	+	1.49543174143753	7	ENSMUST00000155020
chr1	4806396	4808096	1701	+	1.45132490636704	8	ENSMUST00000119612
chr1	4806398	4808098	1701	+	1.43786178107607	9	ENSMUST00000137887
chr1	4806411	4808111	1701	+	1.41219329214475	10	ENSMUST00000115529
chr1	4806418	4808118	1701	+	1.41219329214475	11	ENSMUST00000150971
chr1	4806737	4808437	1701	+	1.46171741778319	12	ENSMUST00000131119
chr1	4835405	4837105	1701	+	NA	13	ENSMUST00000141278
chr1	4856314	4858014	1701	+	1.58979249448124	14	ENSMUST00000081551
chr1	4856538	4858238	1701	+	1.73958654120331	15	ENSMUST00000165720
chr1	4969357	4971057	1701	+	NA	16	ENSMUST00000144339

附录 相关代码

MethylKit

MethylKit Part1:

library(methylKit)
setwd("/home/huangml/WangYunpeng/RRBS/result/TilingWindows")
#与4.0相比,依旧没去除PCR影响,仅仅是窗口化而已(去除PCR影响聚类效果没有明显变好,考虑到后面的数据提取,没有多此一举)
#读入文件
myobj=methRead(list("/home/huangml/WangYunpeng/RRBS/result/P11m414_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P11m415_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P11m416_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m76_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m77_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m78_CpG_filter.txt"),
                 sample.id=list("P11m414","P11m415","P11m416","P3m76","P3m77","P3m78"),
                 assembly="M.musculus",
                 treatment=c(1,1,1,0,0,0),
                 context="CpG"
)


#画出样本甲基化状态图
pdf("P11m414_MethylationStats.pdf")
getMethylationStats(myobj[[1]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P11m415_MethylationStats.pdf")
getMethylationStats(myobj[[2]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P11m416_MethylationStats.pdf")
getMethylationStats(myobj[[3]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m76_MethylationStats.pdf")
getMethylationStats(myobj[[4]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m77_MethylationStats.pdf")
getMethylationStats(myobj[[5]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m78_MethylationStats.pdf")
getMethylationStats(myobj[[6]],plot=TRUE,both.strands=FALSE)
dev.off()

#画出样本甲基化覆盖度图
pdf("P11m414_CoverageStats.pdf")
getCoverageStats(myobj[[1]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P11m415_CoverageStats.pdf")
getCoverageStats(myobj[[2]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P11m416_CoverageStats.pdf")
getCoverageStats(myobj[[3]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m76_CoverageStats.pdf")
getCoverageStats(myobj[[4]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m77_CoverageStats.pdf")
getCoverageStats(myobj[[5]],plot=TRUE,both.strands=FALSE)
dev.off()
pdf("P3m78_CoverageStats.pdf")
getCoverageStats(myobj[[6]],plot=TRUE,both.strands=FALSE)
dev.off()


#过滤数据,减少PCR影响
filtered.myobj=filterByCoverage(myobj,lo.count=10,lo.perc=NULL, hi.count=NULL,hi.perc=99.9)

#将样本聚合在一起
meth=unite(myobj, destrand=FALSE)

#画出样本关联度的图
pdf("P11m-P3mCorrelation.pdf")
getCorrelation(meth,plot=TRUE)
dev.off()

#画出样本聚类的图
pdf("P11m-P3mclusterSamples.pdf")
clusterSamples(meth, dist="correlation", method="ward", plot=TRUE)
dev.off()

#画出样本主成分分析图
pdf("P11m-P3mPCAscreeplot.pdf")
PCASamples(meth, screeplot=TRUE)
dev.off()

#画出样本主成分分析后的图
pdf("P11m-P3mPCAcluster.pdf")
PCASamples(meth)
dev.off()

tiles=tileMethylCounts(myobj,win.size=500,step.size=500)
meth=unite(tiles, destrand=FALSE)

myDiff=calculateDiffMeth(meth,num.cores=12)

# get hyper methylated bases
print("get hyper methylated bases")
myDiff25p.hyper=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hyper")
#
# get hypo methylated bases
print("get hypo methylated bases")
myDiff25p.hypo=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hypo")
#
# get all differentially methylated bases
print("get all differentially methylated bases")
myDiff25p=getMethylDiff(myDiff,difference=25,qvalue=0.01)

#visualize the distribution of hypo/hyper-methylated bases/regions per chromosome
print("visualize the distribution of hypo/hyper-methylated bases/regions per chromosome")
diffMethPerChr(myDiff,plot=FALSE,qvalue.cutoff=0.01, meth.cutoff=25)
pdf("P11m-P3mhypo-hyper-methylatedPerChromosome.pdf")
diffMethPerChr(myDiff,plot=TRUE,qvalue.cutoff=0.01, meth.cutoff=25)
dev.off()

save.image("P11m-P3m.RData")

MethylKit Part2:

library(methylKit)
library(genomation)
setwd("/home/huangml/WangYunpeng/RRBS/result/TilingWindows")
load("P11m-P3m.RData")
#先是读取基因注释信息,用这个注释信息对差异甲基化区域进行注释
gene.obj=readTranscriptFeatures("/home/huangml/WangYunpeng/RRBS/annotation.bed.txt")
diffAnn=annotateWithGeneParts(as(myDiff25p,"GRanges"),gene.obj)
#将差异甲基化最近的基因名输出到文件里
write.csv(getAssociationWithTSS(diffAnn),"P11m-P3m.csv")

getTargetAnnotationStats(diffAnn,percentage=TRUE,precedence=TRUE)

pdf("P11m-P3mAnnotation.pdf")
plotTargetAnnotation(diffAnn,precedence=TRUE, main="differential methylation annotation")
dev.off()


#读取CpG island的注释信息,用这些注释信息来注释我们差异甲基化的区域
cpg.obj=readFeatureFlank("/home/huangml/WangYunpeng/RRBS/cpgi.bed.txt",feature.flank.name=c("CpGi","shores"))

diffCpGann=annotateWithFeatureFlank(as(myDiff25p,"GRanges"),
                                    cpg.obj$CpGi,cpg.obj$shores,
                         feature.name="CpGi",flank.name="shores")

pdf("P11m-P3mCpGisland.pdf")
plotTargetAnnotation(diffCpGann,col=c("green","gray","white"), main="differential methylation annotation")
dev.off()

# 根据之前的注释信息,可以得到起始子区域/CpG island区域的位置,
# 然后下面的方法可以总结这些区域的甲基化信息						 
# promoters=regionCounts(myobj,gene.obj$promoters)
# head(promoters[[1]])

getFeatsWithTargetsStats(diffAnn,percentage=TRUE)

save.image("P11m-P3m-diff.RData")

MethylKit Part3:

library(methylKit)
library(genomation)
setwd("/home/huangml/WangYunpeng/RRBS/result/TilingWindows")
load("P11m-P3m-diff.RData")
# promoters=regionCounts(meth,gene.obj$promoters)
# exons=regionCounts(meth,gene.obj$exons)
# introns=regionCounts(meth,gene.obj$introns)
# TSSes=regionCounts(meth,gene.obj$TSSes)

# write.csv(promoters,"promoters_regionCounts.csv")
# write.csv(exons,"exons_regionCounts.csv")
# write.csv(introns,"introns_regionCounts.csv")
# write.csv(TSSes,"TSSes_regionCounts.csv")

write.csv(gene.obj$promoters,"promoters.csv")
write.csv(gene.obj$exons,"exons.csv")
write.csv(gene.obj$introns,"introns.csv")
write.csv(gene.obj$TSSes,"TSSes.csv")

#write.csv(meth,"P11m-P3m-all.csv")

#write.csv(myDiff25p,"P11m-P3m-myDiff25p.csv")

Diffdataframe=getData(myDiff25p)
index=row.names(Diffdataframe)#meth[index]取回原来每个样本的信息
regionType=data.frame(getMembers(diffAnn))
merge_result=c(getAssociationWithTSS(diffAnn),regionType,myDiff25p,meth[index])
write.csv(merge_result,"P11m-P3m-result.csv")


library(edmr)
library(GenomicRanges)
library(IRanges)
library(mixtools)
library(data.table)
library(methylKit)
setwd("/home/huangml/WangYunpeng/RRBSedmr/liver")
load("P0m-P3m.RData")
setwd("/home/huangml/WangYunpeng/RRBSedmr/liver/edmr")
myDiffFrame<-getData(myDiff)

# fitting the bimodal normal distribution to CpGs distribution
pdf("P0m-P3mDensityCurves.pdf")
myMixmdl=myDiff.to.mixmdl(myDiffFrame, plot=T, main="Density Curves")
dev.off()

# plot cost function and the determined distance cutoff
pdf("P0m-P3mCostfunction.pdf")
plotCost(myMixmdl, main="cost function")
dev.off()

# calculate all DMRs candidate
mydmr=edmr(myDiff, DMC.qvalue = 0.05,num.DMCs = 1,num.CpGs = 1,DMR.methdiff = 5)

# mydmr=edmr(myDiff, mode=1, ACF=TRUE)
result<-DataFrame(mydmr)


library(TxDb.Mmusculus.UCSC.mm10.ensGene)
mm10genes<- genes(TxDb.Mmusculus.UCSC.mm10.ensGene)
geneID<- nearest(mydmr,mm10genes)
geneName<- mm10genes[geneID]@elementMetadata
result<-DataFrame(mydmr,geneName)
write.table(result,file = "P0m-P3m-Edmr-result.txt",quote = FALSE,sep = "\t",row.names = FALSE)

edmr

edmr Part1:

library(methylKit)
setwd("/home/huangml/WangYunpeng/RRBSedmr/liver/")
#进行差异点计算,从而为edmr提供数据
#读入文件
myobj=methRead(list("/home/huangml/WangYunpeng/RRBSnew/result/data/P0m14liver_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBSnew/result/data/P0m15liver_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBSnew/result/data/P0m16liver_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m76_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m77_CpG_filter.txt",
                    "/home/huangml/WangYunpeng/RRBS/result/P3m78_CpG_filter.txt"),
                 sample.id=list("P0m14liver","P0m15liver","P0m16liver","P3m76","P3m77","P3m78"),
                 assembly="M.musculus",
                 treatment=c(1,1,1,0,0,0),
                 context="CpG"
)

#将样本聚合在一起
meth=unite(myobj, destrand=FALSE)

myDiff=calculateDiffMeth(meth,num.cores=12)

# get hyper methylated bases
print("get hyper methylated bases")
myDiff25p.hyper=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hyper")
#
# get hypo methylated bases
print("get hypo methylated bases")
myDiff25p.hypo=getMethylDiff(myDiff,difference=25,qvalue=0.01,type="hypo")
#
# get all differentially methylated bases
print("get all differentially methylated bases")
myDiff25p=getMethylDiff(myDiff,difference=25,qvalue=0.01)

#visualize the distribution of hypo/hyper-methylated bases/regions per chromosome
print("visualize the distribution of hypo/hyper-methylated bases/regions per chromosome")
diffMethPerChr(myDiff,plot=FALSE,qvalue.cutoff=0.01, meth.cutoff=25)
pdf("P0m-P3mhypo-hyper-methylatedPerChromosome.pdf")
diffMethPerChr(myDiff,plot=TRUE,qvalue.cutoff=0.01, meth.cutoff=25)
dev.off()

save.image("P0m-P3m.RData")

library(methylKit)
library(genomation)

#先是读取基因注释信息,用这个注释信息对差异甲基化区域进行注释
gene.obj=readTranscriptFeatures("/home/huangml/WangYunpeng/RRBS/annotation.bed.txt")
diffAnn=annotateWithGeneParts(as(myDiff25p,"GRanges"),gene.obj)

getTargetAnnotationStats(diffAnn,percentage=TRUE,precedence=TRUE)

pdf("P0m-P3mAnnotation.pdf")
plotTargetAnnotation(diffAnn,precedence=TRUE, main="differential methylation annotation")
dev.off()


#读取CpG island的注释信息,用这些注释信息来注释我们差异甲基化的区域
cpg.obj=readFeatureFlank("/home/huangml/WangYunpeng/RRBS/cpgi.bed.txt",feature.flank.name=c("CpGi","shores"))

diffCpGann=annotateWithFeatureFlank(as(myDiff25p,"GRanges"),
                                    cpg.obj$CpGi,cpg.obj$shores,
                         feature.name="CpGi",flank.name="shores")

pdf("P0m-P3mCpGisland.pdf")
plotTargetAnnotation(diffCpGann,col=c("green","gray","white"), main="differential methylation annotation")
dev.off()

# 根据之前的注释信息,可以得到起始子区域/CpG island区域的位置,
# 然后下面的方法可以总结这些区域的甲基化信息						 
# promoters=regionCounts(myobj,gene.obj$promoters)
# head(promoters[[1]])

getFeatsWithTargetsStats(diffAnn,percentage=TRUE)

library(methylKit)
library(genomation)

setwd("/home/huangml/WangYunpeng/RRBSedmr/liver/Meth0")

# 比起原来只有gene.obj注释,多了CpG注释信息
myDiff0p=getMethylDiff(myDiff,difference=0,qvalue=1)
myDiff0p.hyper=getMethylDiff(myDiff,difference=0,qvalue=1,type="hyper")
myDiff0p.hypo=getMethylDiff(myDiff,difference=0,qvalue=1,type="hypo")

#先是读取基因注释信息,用这个注释信息对差异甲基化区域进行注释
gene.obj=readTranscriptFeatures("/home/huangml/WangYunpeng/RRBS/annotation.bed.txt")
diffAnn=annotateWithGeneParts(as(myDiff0p.hyper,"GRanges"),gene.obj)

#读取CpG island的注释信息,用这些注释信息来注释我们差异甲基化的区域
cpg.obj=readFeatureFlank("/home/huangml/WangYunpeng/RRBS/cpgi.bed.txt",feature.flank.name=c("CpGi","shores"))

diffCpGann=annotateWithFeatureFlank(as(myDiff0p.hyper,"GRanges"),
                                    cpg.obj$CpGi,cpg.obj$shores,
                         feature.name="CpGi",flank.name="shores")

CpGType<-data.frame(diffCpGann@members)

Diffdataframe<-getData(myDiff0p.hyper)
index<-row.names(Diffdataframe)#meth[index]取回原来每个样本的信息
regionType<-data.frame(diffAnn@members)
dist.to.TSS<-data.frame(getAssociationWithTSS(diffAnn))
merge_result<-data.frame(dist.to.TSS,regionType,CpGType,myDiff0p.hyper,meth[index])

write.table(merge_result,file = "P0m-P3mhyper-result.txt",quote = FALSE,row.names = FALSE)

# -------------------------------------------------------------------------------

#先是读取基因注释信息,用这个注释信息对差异甲基化区域进行注释
gene.obj=readTranscriptFeatures("/home/huangml/WangYunpeng/RRBS/annotation.bed.txt")
diffAnn=annotateWithGeneParts(as(myDiff0p.hypo,"GRanges"),gene.obj)

#读取CpG island的注释信息,用这些注释信息来注释我们差异甲基化的区域
cpg.obj=readFeatureFlank("/home/huangml/WangYunpeng/RRBS/cpgi.bed.txt",feature.flank.name=c("CpGi","shores"))

diffCpGann=annotateWithFeatureFlank(as(myDiff0p.hypo,"GRanges"),
                                    cpg.obj$CpGi,cpg.obj$shores,
                         feature.name="CpGi",flank.name="shores")

CpGType<-data.frame(diffCpGann@members)

Diffdataframe<-getData(myDiff0p.hypo)
index<-row.names(Diffdataframe)#meth[index]取回原来每个样本的信息
regionType<-data.frame(diffAnn@members)
dist.to.TSS<-data.frame(getAssociationWithTSS(diffAnn))
merge_result<-data.frame(dist.to.TSS,regionType,CpGType,myDiff0p.hypo,meth[index])

write.table(merge_result,file = "P0m-P3mhypo-result.txt",quote = FALSE,row.names = FALSE)

edmr Part2:

library(edmr)
library(GenomicRanges)
library(IRanges)
library(mixtools)
library(data.table)
library(methylKit)
setwd("/home/huangml/WangYunpeng/RRBSedmr/liver")
load("P0m-P3m.RData")
setwd("/home/huangml/WangYunpeng/RRBSedmr/liver/edmr")
myDiffFrame<-getData(myDiff)

# fitting the bimodal normal distribution to CpGs distribution
pdf("P0m-P3mDensityCurves.pdf")
myMixmdl=myDiff.to.mixmdl(myDiffFrame, plot=T, main="Density Curves")
dev.off()

# plot cost function and the determined distance cutoff
pdf("P0m-P3mCostfunction.pdf")
plotCost(myMixmdl, main="cost function")
dev.off()

# calculate all DMRs candidate
mydmr=edmr(myDiff, DMC.qvalue = 0.05,num.DMCs = 1,num.CpGs = 1,DMR.methdiff = 5)

# mydmr=edmr(myDiff, mode=1, ACF=TRUE)
result<-DataFrame(mydmr)


library(TxDb.Mmusculus.UCSC.mm10.ensGene)
mm10genes<- genes(TxDb.Mmusculus.UCSC.mm10.ensGene)
geneID<- nearest(mydmr,mm10genes)
geneName<- mm10genes[geneID]@elementMetadata
result<-DataFrame(mydmr,geneName)
write.table(result,file = "P0m-P3m-Edmr-result.txt",quote = FALSE,sep = "\t",row.names = FALSE)

Promoter甲基化程度分析

Promoter Demo版本

library(TxDb.Mmusculus.UCSC.mm10.ensGene)
txdb_mm10 <- TxDb.Mmusculus.UCSC.mm10.ensGene
trans <- as.data.frame(transcripts(txdb_mm10))
# 取出正链上的基因
transUp <- trans[trans$strand=="+",]
# 取出Tss上游200到下游1500bp内的范围
grPromoterUp <- GRanges(seqnames = transUp$seqnames,strand = "+",
                 ranges = IRanges(start = transUp$start-1500,end = transUp$start+200))
# # 取出负链上的基因
# transDown <- trans[trans$strand=="-",]
# # 取出Tss上游200到下游1500bp内的范围
# grPromoterDown <- GRanges(seqnames = transDown$seqnames,strand = "-",
#                       ranges = IRanges(start = transDown$end-200,end = transDown$end+1500))
grPromoterUp$value <- NaN #value存储每个promoter区域的甲基化程度
wt01Cpg <- read.table("C:/Users/f/Desktop/wt01_CpG_filter.txt",header = TRUE)
grCpG <- GRanges(seqnames = wt01Cpg$chr,
                 ranges = IRanges(start = wt01Cpg$base, width = 1))
grCpG$meth <- wt01Cpg$freqC
grCpG$coverage <- wt01Cpg$coverage
hitObj<- findOverlaps(grPromoterUp,grCpG)
promoterid<- unique(hitObj@from)
for(i in 1:length(promoterid)){
  CpGid<- hitObj[hitObj@from==promoterid[i],]@to
  promoterCpG <- grCpG[CpGid]
  grPromoterUp[promoterid[i]]$value<- weighted.mean(promoterCpG$meth,promoterCpG$coverage) #加权平均值
}
grPromoterUp <- as.data.frame(grPromoterUp)
grPromoterUp <- cbind(grPromoterUp,transUp$tx_id,transUp$tx_name)
write.table(grPromoterUp,"wt01UpStrandPromoterMeth.txt",sep = "\t",quote = FALSE,row.names = FALSE)
#Further :把负链上的基因也做一遍
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