Isothermal amplification method for next-generation sequencing

Abstract

We report an approach for generating immobilized monoclonal templates for next-generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solidsurface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometersized colonies in a single lane of a next-generation sequencing flowchip. An alternative paired-end sequencing method using interstrand DNA photo cross-linking to covalently link the complementary strands of the original templates to the solid surface is also demonstrated.genome | in situ PCR | forming colonies | DNA breathing | polony T he introduction of massively parallel next-generation sequencing (NGS) has revolutionized the biomedical research of human disease and health. The general workflow for NGS involves library generation, colony formation, sequencing, and analysis (1, 2). All NGS technologies, except for single-molecule sequencing platforms, require library amplification to generate many copies of each single library fragment before sequencing. The polymerase colony (polony) technology developed by Church's group was the first reported approach to generating monoclonal colonies by performing PCR on a thin polyacrylamide gel on a microscope slide support, and then covalently attaching the amplified DNA to the polyacrylamide matrix using a 5′ acrydite-modified PCR primer (3). Polony technology was used in digital genotyping, haplotyping (4), and later in sequencing (5).Commercial NGS platforms use two major amplification methods: emulsion PCR (emPCR) with microbeads, which is used by Roche's 454 (6) and Life Technologies' SOLiD (7) and Ion Torrent (8) platforms, and bridge PCR (bPCR) amplification (9-12) on a glass surface, which is used by Illumina's HiSeq/ MiSeq sequencing platforms. Dressman et al. (13) first demonstrated an emPCR approach with superparamagnetic beads in 2003. emPCR requires performing PCR on billions of microbeads, each isolated in its own emulsion droplet, followed by emulsion breakup, template enrichment, and bead deposition before sequencing. bPCR generates monoclonal clusters directly on a solid surface with a minimum of hands-on time. A disadvantage of bPCR is that each of the multiple amplification cycles requires the addition of fresh mixture containing polymerase, followed by a chemical denaturation step, making it cost much more than regular PCR. bPCR also requires a specialized pumping system, such as cBot or a dedicated module built inside the HiSeq/MiSeq system. Another library amplification method developed by Complete Genomics uses rolling-circle replication to amplify small fragments of genomic DNA into DNA nanoballs (14). This approach requires multiple PCR steps to construct a complete circular template before the final amplification step to generate nanoballs.We report here a solid-phase, in situ isothermal PCR approach that generates more than one billion monoclonal colonies in a single lane of a 5500 fl...

Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications

“…The DNA libraries are labeled with barcode sample tags, such as the multiplex identifier (MID) for Roche/454 sequencing, to enable the libraries to be pooled and therefore maximize the sequence output as a multiplex amplicon sequencing step for each sequencing run. After library construction, the DNA fragments are clonally amplified by emulsion PCR with microbeads [4,6,60] or by solid-phase PCR using primers attached to a solid surface [4,61,62] in order to generate sufficient single-stranded DNA molecules and detectable signal for producing sufficiently reliable sequencing data [54]. Roche 454, Life Technologies' SOLiD, and Ion Torrent platforms use emulsion PCR, whereas Illumina's HiSeq/MiSeq platforms use solid-phase PCR [4].…”

“…Roche 454, Life Technologies' SOLiD, and Ion Torrent platforms use emulsion PCR, whereas Illumina's HiSeq/MiSeq platforms use solid-phase PCR [4]. More recently, isothermal PCR amplification on a solid surface of a flow cell [62] was developed for the SOLiD 5500 W series of sequencing machines.…”

“…Previously, sample preparation and amplification was similar to that of Roche 454 sequencing [66]. However, the upgrades to Wildfire chemistry have enabled greater throughput and simpler workflows by replacing beads with direct in situ amplification on FlowChips and paired-end sequencing [62]. The SOLiD 5500 W series sequencing reactions still use fluorescently labeled octamer probes in repeated cycles of annealing and ligation that are interrogated and eventually deciphered in a complex subtractive process using Exact Call Chemistry that has been well described by others [2,36,66].…”

A Computational Framework for DNA Sequencing-Based Microscopy

“…The assignment of barcodes to patches of a surface can be accomplished with surface polony generation methods such as bridge amplification [2], a 2primer rolling circle amplification-based method [11], template walking amplification [13], whereby unique "seed" strands are captured by the surface containing primer strands (Figure 1 A) and then locally amplified in the immediate spatial vicinity of where they landed thus generating a diverse surface of distinct patches of amplified DNA (Figure 1 B). A polony is defined as one of these locally amplified patches of DNA derived from a single seed molecule.…”

Untitled

“…Further, RPA has also been shown to be biased against sequences with high GC content (Hansen et al 2016) as the strand exchange activity of some recombinase proteins has been shown to be inhibited by GC content of 70% (Patil et al 2011). Previous studies have successfully used isothermal amplification on degraded DNA from forensic samples and formalin fixed paraffin embedded (FFPE) tissues (Tate et al 2011;Wang et al 2004), as well as modern sequencing libraries (Jasmine et al 2008;Lou et al 2013;Ma et al 2013;Oyola et al 2012). While, isothermal methods have been also used in the hybridization capture of targets from sequencing libraries made from aDNA (Haak et al 2015), their performance compared to PCR was not evaluated.…”