Noninvasive prenatal diagnosis of monogenic disorders based on direct haplotype phasing through targeted linked-read sequencing

Background Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband’s samples. Methods The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis. Results Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call. Conclusions Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.

Salting-out Protocol for Extracting HMW Genomic DNA from frozen octocorals v1

Salting-out protocol for extracting high-molecular weight (HMW) genomic DNA from frozen octocorals for genomic methods. Suitable for genome sequencing, RAD-seq and other methods that require HMW DNA. Starting material must be alive when flash-frozen in liquid nitrogen and always stored below -80C.

FindingNemo Extraction 2: Phenol-free Method v2

This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using a phenol-free extraction method. A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters. Kit-free, phenol-free protocol is a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads for DNA precipitation matrix. We tested this sub-protocol in human cell line, with input cells of 3 millions but can be varied from 1-5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.

Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle Tribolium and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. Challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction, and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance, and the number of circularized elements found in sequencing results suggest that non-PC methods may be more appropriate for long-read sequencing of metagenomes.

Biodiversity genomics of small metazoans: high quality de novo genomes from single specimens of field-collected and ethanol-preserved springtails

ABSTRACTGenome sequencing of all known eukaryotes on Earth promises unprecedented advances in evolutionary sciences, ecology, systematics and in biodiversity-related applied fields such as environmental management and natural product research. Advances in DNA sequencing technologies make genome sequencing feasible for many non-genetic model species. However, genome sequencing today relies on large quantities of high quality, high molecular weight (HMW) DNA which is mostly obtained from fresh tissues. This is problematic for biodiversity genomics of Metazoa as most species are small and yield minute amounts of DNA. Furthermore, briging living specimens to the lab bench not realistic for the majority of species.Here we overcome those difficulties by sequencing two species of springtails (Collembola) from single specimens preserved in ethanol. We used a newly developed, genome-wide amplification-based protocol to generate PacBio libraries for HiFi long-read sequencing.The assembled genomes were highly continuous. They can be considered complete as we recovered over 95% of BUSCOs. Genome-wide amplification does not seem to bias genome recovery. Presence of almost complete copies of the mitochondrial genome in the nuclear genome were pitfalls for automatic assemblers. The genomes fit well into an existing phylogeny of springtails. A neotype is designated for one of the species, blending genome sequencing and creation of taxonomic references.Our study shows that it is possible to obtain high quality genomes from small, field-preserved sub-millimeter metazoans, thus making their vast diversity accessible to the fields of genomics.

RbAp46/48LIN-53 and HAT-1 are required for initial CENP-AHCP-3 deposition and de novo centromere formation in Caenorhabditis elegans embryos

ABSTRACTForeign DNA microinjected into the Caenorhabditis elegans germline forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Injected linear, short DNA fragments concatemerize into high molecular weight (HMW)-DNA arrays that are visible as punctate DAPI-stained foci in oocytes, which undergo chromatinization and centromerization in embryos. The inner centromere, inner and outer kinetochore components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, assemble onto the nascent ACs during the first mitosis. Yet, due to incomplete DNA replication of the nascent ACs, centromeric proteins are not oriented at the poleward faces of the nascent ACs in mitosis, resulting in lagging ACs. The DNA replication efficiency of ACs improves over several cell cycles. We found that a condensin subunit, SMC-4, but not the replicative helicase component, MCM-2, facilitates de novo CENP-AHCP-3 deposition on nascent ACs. Furthermore, H3K9ac, H4K5ac, and H4K12ac are highly enriched on newly chromatinized ACs. HAT-1 and RbAp46/48LIN-53, which are essential for de novo centromere formation and segregation competency of nascent ACs, also hyperacetylate histone H3 and H4. Different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53, RbAp46/48LIN-53 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs.