A Simple Cost-Effective Method for Whole-Genome Sequencing, Haplotyping, and Assembly

Abstract

In this chapter, we describe single-tube long fragment read (stLFR), a simple preparation method for whole-genome sequencing and physical haplotyping based on the DNA co-barcoding strategy. Similar to LFR, stLFR applies the concept of adding the same barcode to subfragments derived from the same long DNA molecule. However, instead of a 384-well plate, stLFR uses the surface of micron-sized magnetic beads to create millions of virtual compartments in a single reaction tube. This is enabled by a split and pool barcoded bead preparation process capable of generating ~500,000 copies of the same unique barcode, from a library of ~3.6 billion unique barcodes, on each bead. The instruments and devices used in the stLFR process are easily accessible in nearly all standard molecular biology laboratories, and the cost of reagents can be as low as 30 dollars per sample. stLFR libraries can be sequenced by standard second-generation sequencing instruments (e.g., MGI or Illumina devices), and the barcode sharing information enables detection and phasing of all variations, including large structural variations. In addition, stLFR data can be used to scaffold contigs and de novo assemble genomes.

Keywords: Co-barcoding; Combinatorial barcode beads; Diploid de novo assembly; Experimental haplotyping; Long DNA molecules; Phasing; Whole-genome sequencing.