转录组入门（4）：了解参考基因组及基因注释

来源：hoptop1 21,963

准备工作

参考基因组

测序得到的是几百bp的短read，相当于把拼图打散了给你。如果没有参考基因组，从头(de novo)组装等于是重走人类基因组计划的老路，也就是打散了拼图，却不告诉你原来是什么样子，那么任务将会及其艰巨。

还好人类基因组已经组装好了，我们只需要把我们测得序列回贴（mapping)回去，毕竟人与人之间的差距只有不到1%差异, 允许mismatch就行。

因此第一步就是要去UCSC(http://genome.ucsc.edu/index.html)下载hg19参考基因组（文献要求）

UCSC网站

数据存放站点

hg19

基因组文件

不同文件的所包含的数据在该页面有介绍，其中

chromFa.tar.gz - The assembly sequence in one file per chromosome.Repeats from RepeatMasker and Tandem Repeats Finder (with period of 12 or less) are shown in lower case; non-repeating sequence is shown in upper case.

我将数据存放在Windows的F盘的Data文件夹下，用于后续操作

cd /mnt/f/Data
mkdir reference && cd reference
mkdir -p genome/hg19 && cd genome/hg19
nohup wget http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz &
tar -zvf chromFa.tar.gz
cat *.fa > hg19.fa
rm chr*

这个对新手来说，基因组各种版本需要注意，hg19、GRCH37、 ensembl 75这3种基因组版本应该是大家见得比较多的了，国际通用的人类参考基因组，其实他们储存的是同样的fasta序列，只是分别对应着三种国际生物信息学数据库资源收集存储单位，即NCBI，UCSC及ENSEMBL各自发布的基因组信息而已。有一些参考基因组比较小众，存储的序列也不一样，比如BGI做的炎黄基因组，还有DNA双螺旋结构提出者沃森（Watson）的基因组，还有2016年发表在nature上面的号称最完善的韩国人做的基因组。前期我们先不考虑这些小众基因组，主要就下载hg19和hg38，都是UCSC提供的，虽然hg38相比hg19来说，做了很多改进，优点也不少，但因为目前为止很多注释信息都是针对于hg19的坐标系统来的，我们就都下载了，正好自己探究一下。也顺便下载一个小鼠的最新版参考基因组吧，反正比对也就是睡个觉的功夫，顺便分析一下结果，看看比对率是不是很低。

注释信息

然而参考基因组是一部无字天书，要想解读书中的内容，需要额外的注释信息协助。

因此第二步，就是去gencode数据库（http://www.gencodegenes.org/)下载基因组注释文件。

Gencode

关于基因组各种版本对应关系

版本对应

又到了GTF还是GFF3的抉择时刻，简单介绍了一下他们的格式

GTF/GFF3

GTF（General Transfer Format）其实就是GFF2，以Tab分割，分为如下几列

seqname - name of the chromosome or scaffold; chromosome names can be given with or without the 'chr' prefix. Important note: the seqname must be one used within Ensembl, i.e. a standard chromosome name or an Ensembl identifier such as a scaffold ID, without any additional content such as species or assembly. See the example GFF output below.
source - name of the program that generated this feature, or the data source (database or project name)
feature - feature type name, e.g. Gene, Variation, Similarity
start - Start position of the feature, with sequence numbering starting at 1.
end - End position of the feature, with sequence numbering starting at 1.
score - A floating point value.
strand - defined as + (forward) or - (reverse).
frame - One of '0', '1' or '2'. '0' indicates that the first base of the feature is the first base of a codon, '1' that the second base is the first base of a codon, and so on..
attribute - A semicolon-separated list of tag-value pairs, providing additional information about each feature.

而GFF3(General Feature Format)的格式如下

seqid - name of the chromosome or scaffold; chromosome names can be given with or without the 'chr' prefix. Important note: the seq ID must be one used within Ensembl, i.e. a standard chromosome name or an Ensembl identifier such as a scaffold ID, without any additional content such as species or assembly. See the example GFF output below.
source - name of the program that generated this feature, or the data source (database or project name)
type - type of feature. Must be a term or accession from the SOFA sequence ontology
start - Start position of the feature, with sequence numbering starting at 1.
end - End position of the feature, with sequence numbering starting at 1.
score - A floating point value.
strand - defined as + (forward) or - (reverse).
phase - One of '0', '1' or '2'. '0' indicates that the first base of the feature is the first base of a codon, '1' that the second base is the first base of a codon, and so on..
attributes - A semicolon-separated list of tag-value pairs, providing additional information about each feature. Some of these tags are predefined, e.g. ID, Name, Alias, Parent - see the GFF documentation for more details.

看不出来有啥区别，不想纠结就全下载好了。

nohup wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_26/GRCh37_mapping/gencode.v26lift37.annotation.gtf.gz &
nohuop wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_26/GRCh37_mapping/gencode.v26lift37.annotation.gff3.gz &

我们对文字的理解能力远远小于图片，所以下一步需要下载基因组浏览器