外显子组测序ppt课件
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人类基因组的蛋白编码区域大约包含85%的致病突变。
- Choi M, Scholl U I, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing[J]. Proceedings of the National Academy of Sciences, 2009, 106(45): 19096-19101.
2. Yan X J, Xu J, Gu Z H, et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia[J]. Nature genetics, 2011, 43(4): 309-315.
在人类基因中大约有180,000外显子，占人类基因组的1%，约30MB。
-Ng S B, Turner E H, Robertson P D, et al. Targeted capture and massively parallel sequencing of 12 human exomes[J]. Nature, 2009, 461(7261): 272-276.
1. Choi M, Scholl U I, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing[J]. Proceedings of the National Academy of Sciences, 2009, 106(45): 19096-19101.
3. Platforms A. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia[J]. N Engl J Med, 2013, 2013(368): 2059-2074.
4
Coverage rate
Sequencing depth and coverage of the nine paired initial sequencing samples.
5
三、测序平台
Ion Proton™
Illumina HiSeq
6
基于Ion Proton™的外显子测序流程
7
• The bound DNA is isolated using streptavidincoated Dynabeads® paramagnetic beads, and then amplified and purified. The purified, target-enriched sample is then returned to the Ion Torrent system workflow for emulsion PCR, enrichment, and sequencing.
• Exome sequencing results on the Ion Proton™ System using the Ion PI™ Chip and the Ion TargetSeq™ Exome Kit
8
基于Ion Proton™的外显子测序结果
Raw reads
Reads mapped Percent reads mapped
外显子组测序
1
目录
一、外显子测序简介 二、测序深度 三、测序平台 四、数据分析流程 五、数据分析内容 六、后期验证
2
一、外显子测序简介
外显子测序（也称目标外显子组捕获）是指利用序列捕获技术将全 基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法。 是一种选择基因组的编码序列的高效策略，外显子测序相对于基因组重 测序成本较低，对研究已知基因的SNP、Indel等具有较大的优势。
Reads on target
Percent reads on target
ቤተ መጻሕፍቲ ባይዱ
89,782,719 87,156,364 97.1%
Mean depth of coverage
Target bases at 1x
68,899,95
7
Target bases at 10x
79.1%
Target bases at 20x
• The average cover-age of each base in the targeted regions was 100-fold, and 95.3% of these bases were covered sufficiently deeply for variant calling (≥10× cover-age) [2]
3
二、测序深度
• The sensitivity to detect heterozygous variants with 10 reads is 78.6%, but increases to 95.2% at 20x and approximately 100% at 30x and greater.[1]
119x
Type
98.5%
95.3%
92.5%
Number of variants
Concordance with dbSNP135
SNVs
30,095
Heterozygous SNVs 18,031
98.0% 97.1%
Homozygous SNVs 12,046
99.4%
9
• Exome sequencing produced a higher level of coverage for the targeted sequences (mean, 167.50×), slightly increasing our ability to detect mutations with VAFs of less than 10%. [3]
- Choi M, Scholl U I, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing[J]. Proceedings of the National Academy of Sciences, 2009, 106(45): 19096-19101.
2. Yan X J, Xu J, Gu Z H, et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia[J]. Nature genetics, 2011, 43(4): 309-315.
在人类基因中大约有180,000外显子，占人类基因组的1%，约30MB。
-Ng S B, Turner E H, Robertson P D, et al. Targeted capture and massively parallel sequencing of 12 human exomes[J]. Nature, 2009, 461(7261): 272-276.
1. Choi M, Scholl U I, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing[J]. Proceedings of the National Academy of Sciences, 2009, 106(45): 19096-19101.
3. Platforms A. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia[J]. N Engl J Med, 2013, 2013(368): 2059-2074.
4
Coverage rate
Sequencing depth and coverage of the nine paired initial sequencing samples.
5
三、测序平台
Ion Proton™
Illumina HiSeq
6
基于Ion Proton™的外显子测序流程
7
• The bound DNA is isolated using streptavidincoated Dynabeads® paramagnetic beads, and then amplified and purified. The purified, target-enriched sample is then returned to the Ion Torrent system workflow for emulsion PCR, enrichment, and sequencing.
• Exome sequencing results on the Ion Proton™ System using the Ion PI™ Chip and the Ion TargetSeq™ Exome Kit
8
基于Ion Proton™的外显子测序结果
Raw reads
Reads mapped Percent reads mapped
外显子组测序
1
目录
一、外显子测序简介 二、测序深度 三、测序平台 四、数据分析流程 五、数据分析内容 六、后期验证
2
一、外显子测序简介
外显子测序（也称目标外显子组捕获）是指利用序列捕获技术将全 基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法。 是一种选择基因组的编码序列的高效策略，外显子测序相对于基因组重 测序成本较低，对研究已知基因的SNP、Indel等具有较大的优势。
Reads on target
Percent reads on target
ቤተ መጻሕፍቲ ባይዱ
89,782,719 87,156,364 97.1%
Mean depth of coverage
Target bases at 1x
68,899,95
7
Target bases at 10x
79.1%
Target bases at 20x
• The average cover-age of each base in the targeted regions was 100-fold, and 95.3% of these bases were covered sufficiently deeply for variant calling (≥10× cover-age) [2]
3
二、测序深度
• The sensitivity to detect heterozygous variants with 10 reads is 78.6%, but increases to 95.2% at 20x and approximately 100% at 30x and greater.[1]
119x
Type
98.5%
95.3%
92.5%
Number of variants
Concordance with dbSNP135
SNVs
30,095
Heterozygous SNVs 18,031
98.0% 97.1%
Homozygous SNVs 12,046
99.4%
9
• Exome sequencing produced a higher level of coverage for the targeted sequences (mean, 167.50×), slightly increasing our ability to detect mutations with VAFs of less than 10%. [3]