The phosphorothioate bond (S-bond) is a modification used for synthetic oligonucleotides, and PCR primers containing S-bonds show partial or complete tolerance to a range of nucleases, largely depending on the number of inserted S-bonds (, ). In the present study, an alternative short dsDNA fragment assembly method has been developed through the use of T7 exonuclease (T7 gene 6 exonuclease), which has a moderate 5ʹ→3ʹ exodeoxyribonuclease activity ( ). This method, however, is not suitable for assembly of short DNA fragments (<250 bp in length), presumably due to the strong enzymatic activity of T5 exonuclease. The DNA fragments are then annealed on the basis of sequence homology between the cohesive ends (typically 15–80 bp in length), and larger concatenated DNA molecules are generated through gap-filling and ligation with the DNA polymerase and Taq DNA ligase. DNA fragments are partially digested with T5 exonuclease, which catalyses DNA degradation in the 5ʹ to 3ʹ direction, so generating dsDNA with cohesive ends on the 3ʹ-termini. Double-stranded DNA (dsDNA) fragments can be integrated into a larger single molecule through the activities of T5 exonuclease, DNA polymerase and DNA ligase. The homology-based enzymatic DNA fragment assembly (or more commonly termed ‘Gibson assembly’) is a simple DNA manipulation method, widely used in synthetic biology. The DNA molecules prepared for such purposes, however, can be considerably shorter than the potential length of sequencing reads from the current high-throughput sequencing systems. Generation of up to several billion sequencing reads from a single run permits identification of low prevalence sRNAs, accurate determination of sRNA expression levels, detection of functional sequence variation and presence of eDNA. Owing to considerable reductions in both reagent cost and processing time, short-read technologies have become used for not only whole genome shotgun (re-)sequencing, but also other purposes, including small RNA (sRNA) sequencing, molecular inversion probe (MIP)-based mutant detection and characterisation of environmental DNA (eDNA). However, the current Illumina HiSeq platform can generate up to 2 × 250 bp reads, while even longer reads, 2 × 300 bp, can be generated on the MiSeq platform ( ). The read length of an early version of the Illumina (Solexa) platform was only 25–35 bp (2 × 25–35 bp paired-end), considerably shorter than that of earlier Sanger sequencing-based platforms (up to 800–1000 bp). One of the major improvements has been in the length of sequencing reads. Sequencing-by-synthesis technologies (originally by Solexa) have realised the promise of high-throughput sequencing, and a further substantial progress has been achieved since the appearance of the Illumina DNA sequencing platforms based on this method ( ). The utility of this for sequencing of short fragments has consequently been demonstrated. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The results were validated with other genotyping methods. 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. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. 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. 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. 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. 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.
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