Journal: Nucleic Acids Research

Article Title: Highly selective retrieval of accurate DNA utilizing a pool of in situ -replicated DNA from multiple next-generation sequencing platforms

doi: 10.1093/nar/gky016

Figure Lengend Snippet: Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.

Article Snippet: The sequenced picotiter plate was removed from the 454 GS Junior sequencer before the final bleaching step, sealed using an in-house prepared gasket (Figure ), and filled with a PCR mixture of 60 μl 454_fwd primer, 60 μl 454_rev primer, 804 μl dH 2 O, 24 μl dNTPs, 12 μl Phusion DNA polymerase, and 240 μl 5X Phusion HF Buffer (New England BioLabs).

Techniques: In Situ, Next-Generation Sequencing, Sequencing, Microarray, Synthesized