Shinozuka Hiroshi, Sudheesh Shimna, Shinozuka Maiko, Cogan Noel O I
Agriculture Victoria, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University, Bundoora, Victoria, 3083.
School of Applied Systems Biology, La Trobe University, Bundoora, Victoria, 3086, Australia.
Biol Methods Protoc. 2018 Feb 27;3(1):bpy001. doi: 10.1093/biomethods/bpy001. eCollection 2018.
The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57-66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.
当前的Illumina HiSeq和MiSeq平台分别可以生成长度高达2×250 bp和2×300 bp的双端读数。当通过基于目标富集的方法制备DNA测序文库时，这些读数长度可能比感兴趣的基因组区域长得多。基于基于同源性的酶促DNA片段组装方案开发了一种测序文库制备方法，以允许在单次读数内处理多个PCR产物。使用带有8 bp标签的基因座特异性PCR引物扩增目标序列，并使用这些标签，用DNA聚合酶、T7核酸外切酶和T4 DNA连接酶进行基于同源性的酶促DNA组装。因此，短PCR扩增子可以与Illumina平台特有的测序接头一起组装成单个分子。作为概念验证实验，从豌豆基因组DNA模板中获得的、长度为57 - 66 bp且包含可变核苷酸位置的短PCR扩增子在MiSeq平台上进行组装和测序。结果用其他基因分型方法进行了验证。当组装5个PCR扩增子时，每个读数平均成功鉴定出4.3个目标序列（单核苷酸多态性）。因此，这在短片段测序中的实用性得到了证明。
