2. 遺伝学教室について
  3. 遺伝学教室の基盤技術 Short-read NGS




Short-read NGS





図1. 全エクソーム解析データを用いたコピー数解析:11番染色体と14番染色体の不均衡型転座症例


単一遺伝子疾患の遺伝子解析は主に生殖細胞系列のバリアントを対象としています。これらは両親から遺伝したか、受精前の配偶子形成時、あるいは受精卵形成時に起こったバリアント(de novo)であり、患者さんの全身のどの細胞にも存在します(図2)。そのため、精製度が高く、DNAが多く得られて侵襲性の少ない末梢血を用いて解析を行います。しかし、受精後に起こったバリアントである体細胞バリアントによって起こる単一遺伝子疾患も知られています。体細胞バリアントは病変部組織特異的に認めることが多いため、病変部組織からDNAを抽出し、血液由来DNAとの比較によって体細胞バリアントを検出し(図2)、遺伝学的な原因の同定を行います。


図2. 生殖細胞系列バリアント (Germline variant) と体細胞バリアント (Somatic variant) のシークエンスリードの1例。一本のシークエンスリード内に表示されている塩基はバリアントを示している。

Genetic analysis for monogenic diseases using short read next generation sequencer

Short-read next-generation sequencing is an analysis method that is massively parallel sequencing for several hundred base DNA fragments. This method has been commercially available since 2005 and has contributed significantly to human genetics research. Now, it is beginning to be used as a clinical test in Japan, and is being used not only for genetic research, but also for clinical diagnosis and treatment decisions. In our laboratory, we started to use short-read next-generation sequencers in 2009 and have performed whole-exome sequencing (WES) for epilepsy, intellectual disability, multiple congenital anomalies, neurological disorders, and various other diseases. WES data can be used not only to detect single nucleotide substitutions and small insertions and deletions, but also to perform gene copy number analysis (Figure 1) and linkage analysis. From these analyses, we have identified many new disease-causing genes. In our laboratory, the genetic cause of the disease is found by WES in 30-40% of cases, which is similar to other laboratories. In the remaining 60-70% of cases where the cause of the disease is unknown, the other analysis method is required to find the genetic cause.

A case of unbalanced translocation between chromosomes 11 and 14.

Figure 1. Copy number analysis using whole-exome sequencing data: A case of unbalanced translocation between chromosomes 11 and 14.

Somatic variant analysis

The genetic analysis for the monogenic disease usually focuses on germline variants. Germline variants are inherited from either parent or occur at the time of gametogenesis before fertilization or of firtilization (de novo variants). Their variants exist in every cell of the patient’s body (Figure 2). Thus we usually use the DNA derived from peripheral blood, which is high quality and high yield. However, the monogenic disease caused by variants that occur after fertilization, called somatic variants, are also known. Somatic variants often have tissue-specific variants, so DNA is extracted from the lesion tissue and compared with blood-derived DNA to detect tissue-specific variants and identify the causative pathogenic variant (Figure 2).

Nucleotides displayed on sequencing reads indicate variants.

Figure2. Sequencing reads showing a germline variant o r a somatic variant. Nucleotides displayed on sequencing reads indicate variants.