愿天堂没有头痛
短序列比对的呈现和操纵
Lawrence M, Huber W, Pagès H, Aboyoun P, Carlson M, Gentleman R, Morgan M and Carey V (2013). “Software for Computing and Annotating Genomic Ranges.”PLoS Computational Biology, 9. http://dx.doi.org/10.1371/journal.pcbi.1003118,
1. 关于 GenomicAlignments
GenomicAlignments 可以高效储存和操作短序列比对 (short genomic alignments) ，包括 read counting, computing the coverage, junction detection, 以及比对中核苷酸含量的操作。包内有 GAlignments , GAlignmentPairs , GAlignmentsList 三种对象。
2. 访问序列信息
2.1 载入 BAM 文件
从 BAM 文件得到已比对的 reads 和其序列。
这时用到了一个数据包：RNAseqData.HNRNPC.bam.chr14 .
library(GenomicAlignments)
library(RNAseqData.HNRNPC.bam.chr14)
bamfiles <- RNAseqData.HNRNPC.bam.chr14_BAMFILES
names(bamfiles)
# [1] "ERR127306" "ERR127307" "ERR127308" "ERR127309" "ERR127302"
# [6] "ERR127303" "ERR127304" "ERR127305"
调用 Rsamtools 包内的函数 quickBamFlagSummary() 查看 BAM 文件中的序列是单端或双端比对。
library(Rsamtools)
quickBamFlagSummary(bamfiles[1], main.groups.only=TRUE)
# group | nb of | nb of | mean / max
# of | records | unique | records per
# records | in group | QNAMEs | unique QNAME
# All records........................ A | 800484 | 393300 | 2.04 / 10
# o template has single segment.... S | 0 | 0 | NA / NA
# o template has multiple segments. M | 800484 | 393300 | 2.04 / 10
# - first segment.............. F | 400242 | 393300 | 1.02 / 5
# - last segment............... L | 400242 | 393300 | 1.02 / 5
# - other segment.............. O | 0 | 0 | NA / NA
#
# Note that (S, M) is a partitioning of A, and (F, L, O) is a partitioning of M.
# Indentation reflects this.
在利用 readGAlignments() 读取基因组比对前，需要用函数 ScanBamParam() 构建一些参数。
flag1 <- scanBamFlag(isFirstMateRead=TRUE,
isSecondMateRead=FALSE,
isDuplicate=FALSE,
isNotPassingQualityControls=FALSE)
## 作者在这里为了简化后面的流程，只选择了对应于第1个segment的比对序列
param1 <- ScanBamParam(flag=flag1, what="seq")
gal1 <- readGAlignments(bamfiles[1], use.names=TRUE, param=param1)
class(gal1) ## 获得了一个GAlignments对象
# [1] "GAlignments"
# attr(,"package")
# [1] "GenomicAlignments"
gal1
# GAlignments object with 400242 alignments and 1 metadata column:
# seqnames strand cigar qwidth start
# <Rle> <Rle> <character> <integer> <integer>
# ERR127306.26333541 chr14 + 72M 72 19069583
# ERR127306.12926170 chr14 - 72M 72 19363755
# ERR127306.26974899 chr14 + 72M 72 19369799
# ERR127306.4055622 chr14 + 72M 72 19411097
# ERR127306.13938407 chr14 + 72M 72 19411459
# ... ... ... ... ... ...
# ERR127306.10965452 chr14 - 72M 72 106981361
# ERR127306.11919172 chr14 - 72M 72 106986528
# ERR127306.3567919 chr14 + 72M 72 106989680
# ERR127306.21510817 chr14 + 72M 72 106994763
# ERR127306.661203 chr14 - 72M 72 107003171
# end width njunc |
# <integer> <integer> <integer> |
# ERR127306.26333541 19069654 72 0 |
# ERR127306.12926170 19363826 72 0 |
# ERR127306.26974899 19369870 72 0 |
# ERR127306.4055622 19411168 72 0 |
# ERR127306.13938407 19411530 72 0 |
# ... ... ... ... .
# ERR127306.10965452 106981432 72 0 |
# ERR127306.11919172 106986599 72 0 |
# ERR127306.3567919 106989751 72 0 |
# ERR127306.21510817 106994834 72 0 |
# ERR127306.661203 107003242 72 0 |
# seq
# <DNAStringSet>
# ERR127306.26333541 TGAGAATGAT...ATGGCTGCAT
# ERR127306.12926170 CCCCAGGTAT...TGGACACCAG
# ERR127306.26974899 CATAGATGCA...TAACTTACCA
# ERR127306.4055622 TGCTGGTGCA...CAGGAATCAG
# ERR127306.13938407 CAGGAGGTAG...CTGTCTGGTC
# ... ...
# ERR127306.10965452 GGGAGGCCCT...TGGAGAGAAC
# ERR127306.11919172 CCTGAGAGCC...TCACTCTCTG
# ERR127306.3567919 CAACTTTTAT...AGCAGGGAGG
# ERR127306.21510817 CAAAGCTGGA...TTTTGCTGCC
# ERR127306.661203 CATGACTTGA...TACAGTATTT
# -------
# seqinfo: 93 sequences from an unspecified genome
2.2 各类基础访问函数
seqnames(gal1)
# factor-Rle of length 400242 with 1 run
# Lengths: 400242
# Values : chr14
# Levels(93): chr1 chr10 chr11 ... chrUn_gl000249 chrX chrY
head(seqlevels(gal1))
# [1] "chr1" "chr10"
# [3] "chr11" "chr11_gl000202_random"
# [5] "chr12" "chr13"
strand(gal1)
# factor-Rle of length 400242 with 111564 runs
# Lengths: 1 1 15 20 2 2 2 3 1 2 ... 1 3 3 1 2 2 2 2 1
# Values : + - + - + - + - + - ... - + - + - + - + -
# Levels(3): + - *
head(cigar(gal1))
# [1] "72M" "72M" "72M" "72M" "72M" "72M"
head(njunc(gal1))
# [1] 0 0 0 0 0 0
3. 处理已比对核苷酸序列
当由高通量测序实验产生的 reads 被比对到参考基因组后，一般来说人们提出的问题分为两大类: positional only 和 nucleotide-related.
Positional only questions are about the position of the alignments with respect to the reference genome.
Nucleotide-related questions are about the nucleotide content of the alignments.
针对比对的 "nucleotide-related" 问题， GenomicAlignments 提供了不同层次的工具。
3.1 获得原始序列
BAM 格式中的 read 序列是“反向互补”的，当它们与反义链比对时，我们需要将它们重新“反向互补”，得到原始询问序列 (original query sequences).
确定需要被“反向互补”的 reads:
mcols(gal1)$seq
# A DNAStringSet instance of length 400242
# width seq
# [1] 72 TGAGAATGATGATTTCCAATTTCATCC...TGAACTCATCATTTTTTATGGCTGCAT
# [2] 72 CCCCAGGTATACACTGGACTCCAGGTG...GATACACACACTCAAGGTGGACACCAG
# [3] 72 CATAGATGCAAGAATCCTCAATCAAAT...ACAGCACATTAAAAAGATAACTTACCA
# [4] 72 TGCTGGTGCAGGATTTATTCTACTAAG...AATCCACTTTCTTATCTCAGGAATCAG
# [5] 72 CAGGAGGTAGGCTGTGCGTTCAGCAGT...CTGGTTGATCACCTTGACTGTCTGGTC
# ... ... ...
# [400238] 72 GGGAGGCCCTTTATATAACCATCAGGT...CTAATAGGATAAAAGCATGGAGAGAAC
# [400239] 72 CCTGAGAGCCCCTTGCTGTCCTGAGCA...GCCCTCTGGTGTTCTGATCACTCTCTG
# [400240] 72 CAACTTTTATTTCTTAAACACAAGACA...TCTCAGGTGAGCTGTCGAGCAGGGAGG
# [400241] 72 CAAAGCTGGATGTGTCTAGTGTTTTTA...AATAAGAGCATGTGTGGTTTTGCTGCC
# [400242] 72 CATGACTTGATGGCTGGAACAAATACA...AATACTAGCCTTTGCCATACAGTATTT
oqseq1 <- mcols(gal1)$seq
is_on_minus <- as.logical(strand(gal1) == "-")
oqseq1[is_on_minus] <- reverseComplement(oqseq1[is_on_minus])
每个 read 都会被比对不止一次，所以 gal1 中肯定有重复。
is_dup <- duplicated(names(gal1))
table(is_dup)
# is_dup
# FALSE TRUE
# 393300 6942
去重：
oqseq1 <- oqseq1[!is_dup]
length(oqseq1) ## 检查一下
# [1] 393300
length(gal1)
# [1] 400242
3.2 错配、插入缺失标记、缺口 (Mismatches, indels, and gaps )
由于比对过程中是容许一些错配、插入缺失标记、缺口的，所以 mcols(gal1)$seq 中的序列并不是完全与参考基因组匹配的。
"CIGAR" 包含着这些“小错误”出现在比对中的位置信息。
The ‘CIGAR’ (Compact Idiosyncratic Gapped Alignment Report) string is how the SAM/BAM format represents spliced alignments.
For CIGAR =’6M’, this means there are 6 exact matches to the reference.
head(sort(table(cigar(gal1)), decreasing=TRUE))
#
# 72M 35M123N37M 64M316N8M 38M670N34M 18M123N54M 2M131N70M
# 301920 134 134 133 119 96
colSums(cigarOpTable(cigar(gal1))) ## 总览
# M I D N S H P
# 28815928 1496 818 330945002 0 0 0
# = X
# 0 0
table(njunc(gal1)) ## gap的数量
#
# 0 1 2 3
# 303622 94557 2052 11
References
-
An Introduction to the GenomicAlignments Package
-
Working with aligned nucleotides
-
JEFworks: Cigar Strings For Dummies
https://jef.works/blog/2017/03/28/CIGAR-strings-for-*dummies*/
-
Bioinformatics I/O: Java CIGAR Parser for SAM format
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