Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.

Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ∼100 picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

3' Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3'OH ends in DNA or RNA.

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to insert 5' phosphorylated blunt-end double-stranded DNA to DNA breaks at 3'-recessive ends, gaps, or nicks to form a Y-shaped 3'-branch structure. Therefore, this base pairing-independent ligation is termed 3'-branch ligation (3'BL). In an extensive study of optimal ligation conditions, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency to more than 80%. Ligation efficiency was slightly varied between different donor and acceptor sequences. More interestingly, we discovered that T4 DNA ligase efficiently ligated DNA to the 3'-recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, suggesting a ternary complex formation preference of T4 DNA ligase. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important genomic applications, such as 3'-end labelling by adding a universal sequence; directional tagmentation for NGS library construction that achieve theoretical 100% template usage; and targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays.

Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.