根据物种基因组和注释文件，可以编写脚本，提取特殊的序列结构，完成个性化的分析。本文利用Python3提取拟南芥的最长编码序列，并转化为氨基酸序列输出到文件中。
基因结构
-
真核生物和原核生物具有不同的基因结构，首先我们认识一下真核生物的基因结构。接下来我们根据基因结构，提取分析不同结构。
真核生物的基因结构
简并碱基
个别物种的参考基因组中存在特殊的简并碱基，即在基因组某个位置上存在多种碱基类型，一般可以用单个碱基表示。
- 类型
R: A/G
Y: C/T
M: A/C
K: G/T
S: G/C
W: A/T
H: A/T/C
B: G/T/C
V: G/A/C
D: G/A/T
N: A/T/C/G
-
简并碱基的原因
基因组测序样品为多倍体，在同一位点检测到多种碱基信号；测序样品为混合样品，在同一位点检测到多种碱基信号；测序错误； -
如何处理
在拟南芥的基因组中存在的简并碱基，在翻译的时候统一翻译成氨基酸X（参考NCBI的注释文件）。
提取基因组基因最长蛋白编码序列脚本
#------------------------------------------------------------------
#------------------------------------------------------------------
#作者：香波地海
#时间：2019/09/12
#脚本名称：TAIR10.1_sub_longest_cds.py
#脚本功能：根据拟南芥注释文件的features，提取最长编码蛋白序列
#运行模式：python Script.py genome.fasta genome.gff
#返回结果格式：
# >geneid1
# ATGTTTGGGAAACCCTGCGATGCTACGCT
# >geneid2
# ATGCCCGTAGCTAGCGATCGTAGCTAGCTAGCT
#---------------------------------------------------------------------------------------------------
#---------------------------------------------------------------------------------------------------
#举例说明提取过程
#---------------------------------------------------------------------------------------------------
# cds = {}
#如果不是第一个gene信息的话，则处理上个基因里面的多个编码蛋白序列，计算长度提取最长的编码序列；
# cds['gene1'] = {}
# cds['gene1']['pro1'] = [('chr1','+',1,100)]
# cds['gene1']['pro1'] = [('chr1','+',1,100),('chr1','+',150,200),('chr1','+',250,300),('chr1','+',350,400),('chr1','+',450,500),]
# cds['gene1']['pro2'] = [('chr1','+',1,100)]
# cds['gene1']['pro2'] = [('chr1','+',1,100),('chr1','+',120,200),('chr1','+',220,300),('chr1','+',320,400),('chr1','+',420,500),]
# cds['gene2'] = {}
# cds['gene2']['pro3'] = [('chr1','+',1,100)]
# cds['gene2']['pro3'] = [('chr1','+',1,100),('chr1','+',110,200),('chr1','+',210,300),('chr1','+',310,400),('chr1','+',410,500),]
# cds['gene2']['pro4'] = [('chr1','+',1,100)]
# cds['gene2']['pro4'] = [('chr1','+',1,100),('chr1','+',100,200),('chr1','+',200,300),('chr1','+',300,400),('chr1','+',400,500),]
#处理最后一个基因的多个编码蛋白序列，提取最长的蛋白编码序列
#---------------------------------------------------------------------------------------------------
#---------------------------------------------------------------------------------------------------
import sys
import re
from Bio import SeqIO
#---------------------------------------------------------------------------------------------------
#写一个函数readgff(),读取基因组注释文件，返回gene、mrna、exon、cds、longest_cds的字典
#---------------------------------------------------------------------------------------------------
def readgff(gff_file):
'''
'''
g_id = ''
gene = {}
mrna = {}
exon = {}
cds = {}
longest_cds = {}
with open(gff_file) as gff:
for line in gff:
if line.startswith('#'):
continue
else:
line = line.strip()
(chrs,ref,features,start,end,dot,strand,start_point,note)= line.split('\t')
if features == 'gene' :
#-----------------------------------------------------------------------------------
#在读取下一个基因之前，筛选上一个基因的转录本，构建每个基因对应的最长转录本字典
#-----------------------------------------------------------------------------------
if g_id != '':
if cds.get(g_id,None):
temp = {}
for pro, pos in cds[g_id].items():
sum = 0
for i in pos:
sum += int(i[-1]) - int(i[-2]) + 1
temp[pro] = sum
longest_pro = max(temp,key=temp.get)
longest_cds[g_id] = cds[g_id][longest_pro]
#-------------------------------------------------------------------------------
#如果g_id为空值，说明这是注释的第一个基因，那么读取基因ig，构建新的基因字典；如果g_id不是空值，
#那么找到上一个基因的最长转录本之后，记录和存储下一个基因的各种信息
#-------------------------------------------------------------------------------
g_id = note.split(';')[0].replace('ID=','')
gene[g_id] = (chrs,strand,start,end)
cds[g_id] = {}
exon[g_id] = {}
mrna[g_id] = {}
if features == 'mRNA':
r_id = note.split(';')[0].replace('ID=','')
mrna[g_id][r_id] = (chrs,strand,start,end)
if features == 'exon':
r_id = note.split(';')[1].replace('Parent=','')
if exon[g_id].get(r_id,None):
exon[g_id][r_id].append((chrs,strand,start,end))
else:
exon[g_id][r_id] = [(chrs,strand,start,end)]
if features == 'CDS':
c_id = note.split(';')[0].replace('ID=cds-','')
if cds[g_id].get(c_id,None):
cds[g_id][c_id].append((chrs,strand,start,end))
else:
cds[g_id][c_id] = [(chrs,strand,start,end)]
else:
continue
#-----------------------------------------------------------------------------------------------
#分析最后一个基因的最长转录本
#-----------------------------------------------------------------------------------------------
if cds.get(g_id,None):
temp = {}
for pro, pos in cds[g_id].items():
sum = 0
for i in pos:
sum += int(i[-1]) - int(i[-2]) + 1
temp[pro] = sum
longest_pro = max(temp,key=temp.get)
longest_cds[g_id] = cds[g_id][longest_pro]
return gene,mrna,exon,cds,longest_cds
#---------------------------------------------------------------------------------------------------
#定义一个函数用于根据基因位置列表，提取并拼接得到orf的函数sequence(postion)
# longest_cds['gene2'] = [('chr1','+',1,100),('chr1','+',100,200),('chr1','+',200,300),
# ('chr1','+',300,400),('chr1','+',400,500),]
#---------------------------------------------------------------------------------------------------
def sequence(position):
'''
根据函数参数元组position提供的位置参数，提取基因的碱基序列，"+"正链的按照起始位置切片提取，"-"负链的转换成反向互补序列提取，
最后输出dna字符串
5'----ATGGCCATTGTAATGGGCCGCTGAAAGGGTGCCCGATAGCAT----3'
3'----TACCGGTAACATTACCCGGCGACTTTCCCACGGGCTATCGTA----5'
'''
hash = {'A':'T','T':'A','C':'G','G':'C','N':'N'}
scaffold,strand,start,end = position
if strand == '+':
dna = Fa[str(scaffold)][int(start)-1:int(end)].upper()
if strand == '-':
dna1 = Fa[str(scaffold)][int(start)-1:int(end)][::-1].upper()
dna = ''.join([hash[i] for i in dna1])
return dna
#---------------------------------------------------------------------------------------------------
#定义一个函数dna_to_aa()，将核苷酸序列转变成氨基酸序列。输入为dna序列，输出为aa序列。
#---------------------------------------------------------------------------------------------------
def dna_to_aa(dna_seq):
'''
定义密码子与氨基酸对应的字典，其中终止密码子用*表示
'''
aa_dict = {
'TTT':'F', 'TTC':'F', 'TTA':'L', 'TTG':'L', 'CTT':'L', 'CTC':'L', 'CTA':'L', 'CTG':'L', 'ATT':'I', 'ATC':'I','ATA':'I',
'ATG':'M', 'GTT':'V', 'GTC':'V', 'GTA':'V', 'GTG':'V', 'TCT':'S', 'TCC':'S', 'TCA':'S', 'TCG':'S', 'CCT':'P', 'CCC':'P',
'CCA':'P', 'CCG':'P', 'ACT':'T', 'ACC':'T', 'ACA':'T', 'ACG':'T', 'GCT':'A', 'GCC':'A', 'GCA':'A', 'GCG':'A','TAT':'Y',
'TAC':'Y', 'CAT':'H', 'CAC':'H', 'CAA':'Q', 'CAG':'Q', 'AAT':'N', 'AAC':'N', 'AAA':'K', 'AAG':'K', 'GAT':'D', 'GAC':'D',
'GAA':'E', 'GAG':'E', 'TGT':'C', 'TGC':'C', 'TGG':'W', 'CGT':'R', 'CGC':'R','CGA':'R','CGG':'R', 'AGT':'S', 'AGC':'S',
'AGA':'R', 'AGG':'R', 'GGT':'G', 'GGC':'G', 'GGA':'G', 'GGG':'G', 'TGA':'*', 'TAA':'*', 'TAG':'*',
}
#定一个空列表，用来存储翻译完成的氨基酸序列
aa_seq = []
for i in range(0,len(dna_seq)-2,3):
codons = dna_seq[i:i+3]
if aa_dict.get(codons):
aa_seq.append(aa_dict[codons])
else:
aa_seq.append('@')
aa = "".join(aa_seq)
return aa
#---------------------------------------------------------------------------------------------------
#---------------------------------------------------------------------------------------------------
try:
genome = sys.argv[1]
gff = sys.argv[2]
gene,mrna,exon,cds,longest_cds = readgff(gff)
Fa = {rec.id:rec.seq for rec in SeqIO.parse(genome,"fasta")}
print('There are {} genes in the genome.'.format(len(gene)))
print('There are {} genes transcripted mRNA.'.format(len(mrna)))
print('There are {} genes transcripted cds.'.format(len(exon)))
print('There are {} longest proteins produced.'.format(len(longest_cds)))
#---------------------------------------------------------------------------------------------------
#---------------------------------------------------------------------------------------------------
#输出最长转录本
# longest_cds['gene2'] = [('chr1','+',1,100),('chr1','+',100,200),('chr1','+',200,300),('chr1','+',300,400),('chr1','+',400,500),]
except IndexError:
print('please input an gff and fasta file!')
finally:
#---------------------------------------------------------------------------------------------------
#---------------------------------------------------------------------------------------------------
with open("out1.fa",'w') as W:
for name,positions in longest_cds.items():
seq = ''
for p in positions:
seq += sequence(p)
aa = dna_to_aa(seq)
W.write(">" + name + "\n")
W.write(aa + "\n")
print('The programs of {} have completed!'.format(sys.argv[0]))
#------------------------------------------------------------------#------------------------------------------------------------------
#结束了!
#------------------------------------------------------------------#------------------------------------------------------------------
