最近開始hi-c為代表的三維轉(zhuǎn)錄組的學(xué)習(xí)。即由于染色體的高度折疊性，即使同一染色體一維上相距很遠(yuǎn)的elements，也有可能空間距離很近，而產(chǎn)生interaction。
hi-c是目前探索相關(guān)信息的主要方法之一，本篇文獻(xiàn)首次提出hi-c method，并進(jìn)一步發(fā)現(xiàn)了染色體compartment分域方法。
- 文獻(xiàn)：Comprehensive mapping of long range interactions reveals folding principles of the human genome
- PubMed ID：19815776
- GEO：GSE18199
- 關(guān)鍵字：hi-c method；A/B compartment
1、構(gòu)建 hi-c library
如上基本步驟為
- （1）cells are crosslinked with formaldehyde;
DNA與甲醇交聯(lián)，固定DNA（如果DNA間發(fā)生interaction，會(huì)有相關(guān)蛋白將interaction 區(qū)域固定在一起） - （2）DNA is digested with a restriction enzyme that leaves a 5′-overhang;
使用限制內(nèi)切酶切開DNA，產(chǎn)生黏性末端（一端有一段缺口），有interaction的DNA片段會(huì)形成一個(gè)H形； - （3）the 5′-overhang is filled, including a biotinylated residue;
粘性末端缺口補(bǔ)平（包含biotinylated，如上圖是那個(gè)紫色的標(biāo)記） - （4）the resulting blunt-end fragments are ligated ;
H兩端分別連接，成θ形 - （5）A Hi-C library is created by shearing the DNA and selecting the biotin-containing fragments with streptavidin beads.
DNA打斷碎片，富集、純化含有biotinylated的片段，最終形成Hi-c library，進(jìn)行測(cè)序。 - （6）parallel DNA sequencing, producing a catalog of interacting fragments
將上述文庫(kù)進(jìn)行雙端測(cè)序，以biotin為分隔成一組read pairs（read1、read2）分別比對(duì)到基因組兩個(gè)位置 A、B
位置A、B即是染色體上的一段區(qū)域。單位長(zhǎng)度即為bin 或者說resolution
bin越短 可匹配的越精確，但同時(shí)匹配的reads 數(shù)就減少了；
bin越長(zhǎng) 匹配到的就越寬泛，但能夠匹配到更多的reads ；
- 測(cè)序結(jié)果表明共有8.4M read pairs total。其中6.7 million corresponded to long-range contacts between segments greater than >20Kb apart.即六百萬+的read pairs 相隔20KB長(zhǎng)度以上，說明許多一維相距很遠(yuǎn)的bin，但空間距離很近。
2、genome-wide contact matrix M Heatmap
- 目的：將上述測(cè)序比對(duì)結(jié)果可視化。
- 矩陣格子：人染色體長(zhǎng)度一般有100~200Mb，文獻(xiàn)中用到的chr14長(zhǎng)度為104M，bin設(shè)置為1Mb。
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橫、縱軸為相同的染色體長(zhǎng)度，以bin長(zhǎng)度（1mb）為軸刻度單位。因此對(duì)應(yīng)的contact matrix M就是104*104大小的矩陣。（如下圖）
2-1 - Mij to be the number of ligation products between locusi and locus j (SOM).
舉例來說，假如M(2,8)=10就表示reads pairs分別匹配到染色體第2個(gè)bin與第8個(gè)bin的數(shù)目為10；再轉(zhuǎn)換為對(duì)應(yīng)熱圖相應(yīng)的顏色等級(jí)。 - This matrix reflects an ensemble average of the interactions present inthe original sample of cells;
- It can be visually represented as a heatmap, with intensity indicating contact frequency.
2-2
如上討論的情況是pairs 的read1，read2均比對(duì)到同一條染色體的情況，稱為
Cis interaction
。而Trans interaction就是指read pairs分別比對(duì)到不同染色體的情況。
3、average intrachromosomal contact probability
- 疑問：如何計(jì)算？（基于1D component？）
- 概念：I n(s) for pairs of loci separated by a genomic distance s onchromosome n.
指在染色體n上，平均相距長(zhǎng)度為s（一維距離）的兩個(gè)position contact probability - 結(jié)果，如下圖：從上往下依次代表染色體1內(nèi)部、染色體1與10、染色體與其余所有染色體平均、染色體1與21。
-
結(jié)論
（1）contact probability decreases monotonically on every chromosome；就是說一般距離近，contact probability（interaction）作用強(qiáng)
（2）chromosome territories.即使一條染色體上相距很遠(yuǎn)（超過200mb），其contact probability也比兩條染色體里的任意兩個(gè)position 高得多
3-2
4、compartment
4.1、observed contact matrix M Heatmap
就是我們?cè)诘诙c(diǎn)討論的內(nèi)容
4.2、nomalization：observed/expected
- 為了校正 sequence proximity strongly influences contact probability，將raw data進(jìn)行標(biāo)準(zhǔn)化；
- 標(biāo)準(zhǔn)化方法：dividing each entry in the contact matrix by the genome-wide average contact probability for loci at that genomic distance （上述第三點(diǎn)）.
-
結(jié)果如下：分為兩大類，紅色的大于1，藍(lán)色的小于1
The normalized matrix shows many large blocks of enriched and depleted interactions generating a ‘plaid’ pattern（格子圖案）
4-2
4.3、correlation matrix C
- If two loci (here 1 Mb regions) are nearby in space, we reasoned that they will share neighbors and have correlated interaction profiles. （未太理解這句話）
-
轉(zhuǎn)換為相關(guān)矩陣correlation matrix C
Cij is the Person correlation between the i row and j column of M which dramatically sharpened the plaid pattern（如下圖）
4-3 - The plaid pattern suggests that each chromosome can be decomposed into two sets of loci(arbitrarily labeled A and B) such that contacts within each set are enriched and contacts between sets are depleted.
即表示將染色體分為兩個(gè)區(qū)域，區(qū)域內(nèi)的bins interaction 明顯高于 區(qū)域間的interacton
4.4、pca的第一pc
- 對(duì)相關(guān)矩陣進(jìn)行主成分分析，一般來說, the first principal component (PC) clearly corresponded to the plaid pattern (positive values defining one set, negative values the other)
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The entries of the PC vector reflected the sharp transitions from compartment to compartment observed within the plaid heatmaps.
4-4
4.5 compartment A B
- The Hi-C data imply that regions tend be closer in space if they belong to the same
compartment. - 但是如上G圖以及文章其它證據(jù)表明 compartment A compartment A is more closely associated with open, accessible, actively transcribed chromatin. 相對(duì)來說compartment B closed chromatin domains ,相對(duì)來說表達(dá)不活躍。
