Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Detection and quantification of mitochondrial DNA deletions from next-generation sequence data.

BACKGROUND: Chromosomal deletions represent an important class of human genetic variation. Various methods have been developed to mine "next-generation" sequencing (NGS) data to detect deletions and quantify their clonal abundances. These methods have focused almost exclusively on the nuclear genome, ignoring the mitochondrial chromosome (mtDNA). Detecting mtDNA deletions requires special care. First, the chromosome's relatively small size (16,569 bp) necessitates the ability to detect extremely focal events. Second, the chromosome can be present at thousands of copies in a single cell (in contrast to two copies of nuclear chromosomes), and mtDNA deletions may be present on only a very small percentage of chromosomes. Here we present a method, termed MitoDel, to detect mtDNA deletions from NGS data. RESULTS: We validate the method on simulated and real data, and show that MitoDel can detect novel and previously-reported mtDNA deletions. We establish that MitoDel can find deletions such as the "common deletion" at heteroplasmy levels well below 1%. CONCLUSIONS: MitoDel is a tool for detecting large mitochondrial deletions at low heteroplasmy levels. The tool can be downloaded at http://mendel.gene.cwru.edu/laframboiselab/ .

Heteroplasmic shifts in tumor mitochondrial genomes reveal tissue-specific signals of relaxed and positive selection.

Although mitochondrial genomes (mtDNA) accumulate elevated levels of mutations in cancer cells, the origin and functional impact of these mutations remain controversial. Here, we queried whole-genome sequence data from 1,916 patients across 24 cancer types to characterize patterns of mtDNA mutations and elucidate the selective constraints driving their fate. Given that mitochondrial genomes are polyploid, cells with advantageous levels of mtDNA mutations can be selected for depending on their cellular environment. Therefore, we tracked changes in per-cell abundances of mtDNA mutations from normal to tumor cells in the same patient. Tumor mitochondrial genomes show distinct mutational patterns and are disproportionately enriched for protein-altering changes. Moreover, protein-altering mtDNA variants that are initially present at low frequencies in normal cells preferentially expand in the altered tumor environment, suggesting selective advantage. We also perform these analyses with attention to the cancer's tissue of origin, which revealed tissue-specific differences in selective signals. The mitochondrial genomes in renal chromophobe and thyroid cancers show particularly strong signals of positive selection, indicated by higher proportions and per-cell abundances of truncating variants. Dramatic tumor- and tissue-specific variations in selective pressures suggest that cancer cells with advantageous levels of damaged mitochondrial genomes will selectively proliferate to facilitate the tumorigenic process.

Correction: PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0139253.].

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.