Hybrid genome assembly

Single molecule real time sequencing

Transcriptome

Barcode

DNA sequencer

Biology

Gene

Illumina dye sequencing

Operating system

Deep sequencing

Genomic library

Gene expression

Genome

Human genome

Whole genome sequencing

Sequence assembly

Massive parallel sequencing

Reference genome

Computer science

DNA sequencing

Computational biology

Base sequence

Sequencing by hybridization

Genetics

DNA nanoball sequencing

Genome Research

Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly