<scp>MIG</scp> ‐seq2: Multiplexed Inter‐Simple Sequence Repeat Genotyping by Sequencing With Dual‐Unique Indices

ABSTRACT Genome‐wide DNA analysis using high‐throughput sequencing is a powerful tool for a wide range of genetic studies. Multiplexed inter‐simple sequence repeat (ISSR) genotyping by sequencing (MIG‐seq) is an effective method that starts with a first polymerase chain reaction (PCR) to amplify genome‐wide regions, followed by a second PCR to add sequencing adaptors and indices. Because the original protocol was designed for a relatively low‐throughput sequencing platform at the time, the primary limitation was the restricted number of distinguishable samples that could be achieved using unique indices. To overcome this limitation, this study aims to adapt the method to recent advances in ultrahigh‐throughput sequencing technology; the second PCR primers were modified to differentiate more than 1344 samples with higher accuracy using dual‐unique indices. Additionally, the tail sequences of the first PCR primers were improved to reduce the number of low‐quality sequences. By combining these two modifications, we developed a new method, MIG‐seq with dual unique indices (MIG‐seq2). Moreover, an optional first PCR primer set, the degenerate oligonucleotide primer MIG‐seq2 (dpMIG‐seq2), was designed to amplify additional regions, mainly in samples with low genetic diversity. To demonstrate the effectiveness of this improved method, a simple phylogenetic analysis was conducted on two closely related species, Plantago hakusanensis and P lantago asiatica var. densiuscula . The resulting phylogenetic trees clearly showed separate clustering of the two species and their intraspecific populations with high reproducibility across analyses using MIG‐seq, MIG‐seq2, and dpMIG‐seq2. This updated version of MIG‐seq is expected to support various genetic studies that utilize high‐throughput genotyping.