一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法与流程

技术特征：
1.一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统，其特征在于，包括：微生物序列参考库模块，菌株耐药参考库模块，病原微生物鉴定模块，突变检测模块和耐药数据库注释模块；所述微生物序列参考库模块为通过多个数据库的微生物rrna序列构建微生物序列参考库，对序列进行过滤，形成高可信度的微生物序列参考库；所述病原微生物鉴定模块包括：测序数据样品拆分模块，数据质控处理模块，比对分析模块和物种分类模块；所述测序数据样品拆分模块为：通过guppy软件将由测序仪产生的fast5格式文件转换成fastq格式，每个样品都具有独特的barcode序列，每条序列的前一段序列就是barcode序列，将这段序列与测序仪提供的barcode序列库进行比对，将比对分数大于80的序列视为可信的分配；所述数据质控处理模块为：采用nanofilt软件进行质控，每条序列都有一个平均测序数据质量值q，若q值小于7的序列为不合格序列，将被删除；所述比对分析模块为：将质控处理后的序列采用minimap2进行快速比对，计算reads 的identity和coverage，筛选identity 大于90%且coverage大于85%的结果；所述物种分类模块为：将序列与微生物序列参考库进行比对，选择比对分数最高、identity大于90%且coverage大于85%的记录作为该序列的最佳比对记录；将最佳比对记录id与物种谱系数据库进行匹配，获取物种分类结果；所述突变检测模块为对来源于菌株的reads采用minimap2比对到菌株参考基因组，采用varscan2检测基因突变位点，获得bam文件，利用samtools对bam文件进行排序，使用samtools mpileup提取mpileup文件，再使用varscan2对mpileup文件进行处理，获得突变结果并以vcf格式保存，然后使用snpeff对突变结果进行基因注释；所述菌株耐药参考库模块为以card数据库结合文献报道的菌株耐药位点信息整合成自建菌株耐药参考库；所述耐药数据库注释模块为对基因突变位点进行注释，获取该突变位点对应的耐药药物名称。2.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统，其特征在于，所述微生物序列参考库模块中的多个数据库包括：ncbi 16s rrna、18s rrna、its 、silva和unite数据库。3.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统，其特征在于，所述微生物序列参考库模块中对序列进行过滤的具体方法包括：步骤a：将来源不同的序列合并成一个综合数据库，并剔除重复序列；步骤b：筛选有文献支持的序列作为高可信度参考序列。4.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统，其特征在于，计算reads 的identity和coverage的方法为： minimap2比对结果中第13列含有前缀de:f:，该列值加上identity等于1；minimap2比对结果第2列代表序列长度，第3列代表比对其实位置，第4列代表比对终止位置，将第4列与第3列的差值除以第2列等于coverage。5.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系
统，其特征在于，所述菌株参考基因组为结核分枝杆菌参考基因组；所述结核分枝杆菌参考基因组为mycobacterium tuberculosis h37rv，ncbi登录号为nc_000962.3。6.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统，其特征在于，所述自建菌株耐药参考库为自建结核分枝杆菌耐药参考库，所述自建结核分枝杆菌耐药参考库包括如下内容：gene
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mutation drug ahpc
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isoniazid
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ahpc
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c
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isoniazid inha
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isoniazid katg
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isoniazid katg
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isoniazid katg
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ꢀꢀꢀꢀ
n508d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
l147p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
n660d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a312p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s383p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
h97r
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀ
r489s
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
m420t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d387h
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g123e
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g299s
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d419h
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
m176t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
r632c
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d542h
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
y64s
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
y95c
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀ
p131t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a139p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d142g
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a162v
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g269d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
t306p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
r385w
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d387g
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
t394m
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q439p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
f483l
ꢀꢀ
isoniazid katg
ꢀꢀꢀ
a541d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g32d
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
v47e
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d194g
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a256t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
t667p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
m624v
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a717p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
i335t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q127p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q352e
ꢀꢀ
isoniazid katg
ꢀꢀꢀ
y98c
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a379t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g269r
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a431v
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d194y
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s17t
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a93t
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d36e
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a110v
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a139v
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g300w
ꢀꢀ
isoniazid katg
ꢀꢀꢀ
h276m
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q295h
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s17n
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g19d
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q224e
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a243s
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
a550d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s140n
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g279d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g285d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g316d
ꢀꢀ
isoniazid katg
ꢀꢀꢀ
s457i
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g593d
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d63e
ꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
n133t
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
p232s
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
q127e
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
i317v
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315r
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315n
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
w191r
ꢀꢀ
isoniazid katg
ꢀꢀꢀ
t667p
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
v469l
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
r463l
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
d735n
ꢀꢀꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
g629s
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
l587m
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315t
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315r
ꢀꢀ
isoniazid katg
ꢀꢀꢀꢀ
s315g
ꢀꢀ
isoniazid pnca
ꢀꢀꢀꢀ
d63g
ꢀꢀꢀ
pyrazinamide pnca q10k
ꢀꢀꢀꢀ
pyrazinamide pnca t47s
ꢀꢀꢀ
pyrazinamide pnca d49v
ꢀꢀꢀ
pyrazinamide pnca s66p
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
k96e
ꢀꢀꢀ
pyrazinamide pnca a102t
ꢀꢀ
pyrazinamide pnca a102v
ꢀꢀ
pyrazinamide pnca l116r
ꢀꢀꢀꢀ
pyrazinamide pnca v125d
ꢀꢀ
pyrazinamide pnca i133t
ꢀꢀ
pyrazinamide pnca q141p
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
r154g
ꢀꢀ
pyrazinamide pnca t160p
ꢀꢀ
pyrazinamide pnca a161p
ꢀꢀ
pyrazinamide pnca d12a
ꢀꢀꢀꢀ
pyrazinamide pnca d12n
ꢀꢀꢀ
pyrazinamide pnca h51q
ꢀꢀꢀ
pyrazinamide pnca a171p
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
h57d
ꢀꢀꢀ
pyrazinamide pnca w68l
ꢀꢀꢀ
pyrazinamide pnca v139a
ꢀꢀ
pyrazinamide pnca r121p
ꢀꢀꢀꢀ
pyrazinamide pnca h137r
ꢀꢀ
pyrazinamide pnca w119r
ꢀꢀ
pyrazinamide pnca f94p
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
t76p
ꢀꢀꢀ
pyrazinamide pnca l19p
ꢀꢀꢀ
pyrazinamide pnca s104c
ꢀꢀ
pyrazinamide pnca i90s
ꢀꢀꢀꢀ
pyrazinamide pnca c14r
ꢀꢀꢀ
pyrazinamide pnca c14y
ꢀꢀꢀ
pyrazinamide pnca g23v
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
v45g
ꢀꢀꢀ
pyrazinamide pnca a46e
ꢀꢀꢀ
pyrazinamide pnca d49a
ꢀꢀꢀ
pyrazinamide pnca f58l
ꢀꢀꢀꢀ
pyrazinamide pnca y68d
ꢀꢀꢀ
pyrazinamide pnca s67p
ꢀꢀꢀ
pyrazinamide pnca w68r
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
w68g
ꢀꢀꢀ
pyrazinamide pnca w68s
ꢀꢀꢀ
pyrazinamide pnca t76i
ꢀꢀꢀ
pyrazinamide pnca g78d
ꢀꢀꢀꢀ
pyrazinamide pnca l85r
ꢀꢀꢀ
pyrazinamide pnca k96q
ꢀꢀꢀ
pyrazinamide pnca y103h
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
y103s
ꢀꢀ
pyrazinamide pnca t135p
ꢀꢀ
pyrazinamide pnca d136n
ꢀꢀ
pyrazinamide pnca d136y
ꢀꢀꢀꢀ
pyrazinamide pnca v139m
ꢀꢀ
pyrazinamide pnca a146t
ꢀꢀ
pyrazinamide pnca v155g
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
l159r
ꢀꢀ
pyrazinamide pnca l172p
ꢀꢀ
pyrazinamide pnca v180f
ꢀꢀ
pyrazinamide pnca l172a
ꢀꢀꢀꢀ
pyrazinamide pnca a171t
ꢀꢀ
pyrazinamide pnca h51y
ꢀꢀꢀ
pyrazinamide pnca p62h
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
a171v
ꢀꢀ
pyrazinamide pnca d136h
ꢀꢀ
pyrazinamide pnca l27p
ꢀꢀꢀ
pyrazinamide pnca p54l
ꢀꢀꢀꢀ
pyrazinamide pnca c72w
ꢀꢀꢀ
pyrazinamide pnca m175v
ꢀꢀ
pyrazinamide pnca h59d
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
a46v
ꢀꢀꢀ
pyrazinamide pnca d53a
ꢀꢀꢀ
pyrazinamide pnca h71e
ꢀꢀꢀ
pyrazinamide pnca h82r
ꢀꢀꢀꢀ
pyrazinamide pnca g97s
ꢀꢀꢀ
pyrazinamide pnca a134v
ꢀꢀ
pyrazinamide pnca c138s
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
t168n
ꢀꢀ
pyrazinamide pnca t47p
ꢀꢀꢀ
pyrazinamide pnca a121p
ꢀꢀ
pyrazinamide pnca y34s
ꢀꢀꢀꢀ
pyrazinamide pnca g17d
ꢀꢀꢀ
pyrazinamide pnca p54t
ꢀꢀꢀ
pyrazinamide pnca a140s
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
t47a
ꢀꢀꢀ
pyrazinamide pnca d12e
ꢀꢀꢀ
pyrazinamide pnca w68c
ꢀꢀꢀ
pyrazinamide pnca t142m
ꢀꢀꢀꢀ
pyrazinamide pnca t142k
ꢀꢀ
pyrazinamide pnca v139l
ꢀꢀ
pyrazinamide pnca c138y
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
h137p
ꢀꢀ
pyrazinamide pnca g132s
ꢀꢀ
pyrazinamide pnca n118t
ꢀꢀ
pyrazinamide pnca k96n
ꢀꢀꢀꢀ
pyrazinamide pnca l85p
ꢀꢀꢀ
pyrazinamide pnca c72r
ꢀꢀꢀ
pyrazinamide pnca p69r
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
y41h
ꢀꢀꢀ
pyrazinamide pnca a26g
ꢀꢀꢀ
pyrazinamide pnca y34d
ꢀꢀꢀ
pyrazinamide pnca d49n
ꢀꢀꢀꢀ
pyrazinamide pnca d49g
ꢀꢀꢀ
pyrazinamide pnca h51r
ꢀꢀꢀ
pyrazinamide pnca p54l
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
h57d
ꢀꢀꢀ
pyrazinamide pnca h57r
ꢀꢀꢀ
pyrazinamide pnca f58s
ꢀꢀꢀ
pyrazinamide pnca p62s
ꢀꢀꢀꢀ
pyrazinamide pnca s67p
ꢀꢀꢀ
pyrazinamide pnca w68r
ꢀꢀꢀ
pyrazinamide pnca w68g
ꢀꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
p69r
ꢀꢀꢀ
pyrazinamide pnca h71r
ꢀꢀꢀ
pyrazinamide pnca t76p
ꢀꢀꢀ
pyrazinamide pnca h82r
ꢀꢀꢀꢀ
pyrazinamide pnca l85r
ꢀꢀꢀ
pyrazinamide pnca g97s
ꢀꢀꢀ
pyrazinamide pnca t100p
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
q10p
ꢀꢀꢀ
pyrazinamide pnca q10r
ꢀꢀꢀ
pyrazinamide pnca l116r
ꢀꢀ
pyrazinamide pnca d12a
ꢀꢀꢀꢀ
pyrazinamide pnca v128g
ꢀꢀ
pyrazinamide pnca i133t
ꢀꢀ
pyrazinamide pnca t135p
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
d136y
ꢀꢀ
pyrazinamide pnca c14r
ꢀꢀꢀ
pyrazinamide pnca c138r
ꢀꢀ
pyrazinamide pnca v139g
ꢀꢀꢀꢀ
pyrazinamide pnca q141p
ꢀꢀ
pyrazinamide pnca a146e
ꢀꢀ
pyrazinamide pnca a146v
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
l151s
ꢀꢀ
pyrazinamide pnca m175v
ꢀꢀ
pyrazinamide pnca v180f
ꢀꢀ
pyrazinamide pnca v180a
ꢀꢀꢀꢀ
pyrazinamide pnca v180g
ꢀꢀ
pyrazinamide pnca l182s
ꢀꢀ
pyrazinamide pnca l182s
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
e174g
ꢀꢀ
pyrazinamide pnca l72p
ꢀꢀꢀ
pyrazinamide pnca a161g
ꢀꢀ
pyrazinamide pnca v157g
ꢀꢀꢀꢀ
pyrazinamide pnca r157w
ꢀꢀ
pyrazinamide pnca t100p
ꢀꢀ
pyrazinamide pnca r140g
ꢀꢀ
pyrazinamide pnca
ꢀꢀꢀꢀ
c138w
ꢀꢀ
pyrazinamide pnca k96m
ꢀꢀꢀ
pyrazinamide pnca s164p
ꢀꢀ
pyrazinamide pnca h57q
ꢀꢀꢀꢀ
pyrazinamide pnca g17s
ꢀꢀꢀ
pyrazinamide rpob s531l
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
n518t
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
s522q
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
k527n
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
r528h
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s512r
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
l521m
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526g
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
f505l
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
s574l
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
g507s
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
g507d
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
t508p
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
t508a
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
t508h
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
q513d
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
q513e
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s456w
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
d516h
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
n519k
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
g523a
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526f
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526q
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
s531f
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s531c
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s512t
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
l511r
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s512i
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526n
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
r529q
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
h526t
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
r528p
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
l524s
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
q517l
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s509q
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s509r
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
e672d
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
h526e
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
l511v
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
l533p
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
d516v
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
t508n
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s512n
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
p520t
ꢀꢀ
rifampicin rpob
ꢀꢀꢀꢀ
s531w
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
s531g
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526d
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
h526s
ꢀꢀꢀꢀ
rifampicin rpob
ꢀꢀꢀ
k527q
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
d516t
ꢀꢀ
rifampicin rpob
ꢀꢀꢀ
d516y
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rifampicin rpob
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streptomycin。7.根据权利要求1所述的一种基于nanopore测序仪进行结核分枝杆菌耐药检测分析系统的构建方法，其特征在于，步骤一，构建用于nanopore测序数据比对的微生物序列参考库：利用多个数据库的微生物rrna序列，构建微生物序列参考库，对序列进行过滤得到高可信度的微生物序列参考库；步骤二，测序待测样品，将测序reads比对到微生物序列参考库并鉴定病原微生物，病原微生物鉴定通过测序数据样品拆分、数据质控处理、比对分析和物种分类对测序数据进行分析；所述测序数据样品拆分的方法具体包括如下步骤：通过guppy软件将由测序仪产生的fast5格式文件转换成fastq格式，找到样品的barcode序列，将这段序列与测序仪提供的barcode序列库进行比对，将比对分数大于80的序列视为可信的分配；所述barcode序列指：每个样品都具有独特的barcode序列，每条序列的前一段序列就是barcode序列；所述数据质控处理的方法为：采用nanofilt软件进行质控，每条序列都有一个平均测序数据质量值q，q值小于7的序列为不合格序列；所述比对分析的方法为：将质控处理后的序列采用minimap2进行快速比对，计算reads 的identity和coverage，筛选identity 大于90%且coverage大于85%作为比对质量高的结果；所述物种分类的方法为：将序列与微生物序列参考库进行比对，选择比对分数最高、identity大于90%且coverage大于85%的记录作为该序列的最佳比对记录；然后将最佳比对记录id与物种谱系数据库进行匹配，获取物种分类结果；步骤三，将来源于菌株的序列比对到菌株参考基因组并鉴定基因突变：将来源于菌株样品序列采用minimap2比对到菌株参考基因组，获得bam文件；利用samtools对bam文件进行排序，使用samtools mpileup提取mpileup文件，再使用varscan2对mpileup文件进行处理，获得突变结果并以vcf格式保存，然后使用snpeff对突变结果进行基因注释；步骤四，以card数据库结合文献报道的菌株耐药位点信息整合成自建菌株耐药参考库；步骤五，将基因突变位点与耐药数据库中的突变位点进行匹配关联，获取基因突变位点对应的耐药药物名称。

技术总结
本发明公开了一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法，属于基因检测分析领域，系统包括：微生物序列参考库模块、结核分枝杆菌耐药参考库模块、病原微生物鉴定模块、突变检测模块和耐药数据库注释模块；基于该系统的构建方法包括：构建数据比对的微生物序列参考库，将测序数据比对到微生物序列参考库并鉴定病原微生物，构建用于突变注释的结核分枝杆菌耐药参考库，将来源于结核分枝杆菌的序列比对到结核分枝杆菌参考基因组并鉴定基因突变，将结核分枝杆菌的基因突变位点与结核分枝杆菌耐药参考库关联分析突变位点的耐药性质；本系统能够快速鉴定病原微生物和检测结核分枝杆菌耐药性质，且灵敏度和特异性高。度和特异性高。度和特异性高。


技术研发人员：谷红仓 谢长校 徐振宇 王云飞 车仙荣
受保护的技术使用者：杭州圣庭医疗科技有限公司
技术研发日：2021.06.17
技术公布日：2021/11/19