Construction of Strand-seq libraries in open nanoliter arrays

Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

Background: CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

Background: CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor

Comprehensive molecular portraits of human breast tumours

This Article from the Cancer Genome Atlas consortium describes a multifaceted analysis of primary breast cancers in 825 people. Exome sequencing, copy number variation, DNA methylation, messenger RNA arrays, microRNA sequencing and proteomic analyses were performed and integrated to shed light on breast-cancer heterogeneity. Just three genes — TP53, PIK3CA and GATA3 — are mutated at greater than 10% frequency across all breast cancers. Many subtype-associated and novel mutations were identified, as well as two breast-cancer subgroups with specific signalling-pathway signatures. The analyses also suggest that much of the clinically observable plasticity and heterogeneity occurs within, and not across, the major subtypes of breast cancer

A combined scanning tunneling microscope and scanning electron microscope

A scanning tunneling microscope (STM) was developed to work in conjunction with a Hitachi S-4100 field emission scanning electron microscope (SEM). To achieve the necessary five degrees of freedom for sample and probe movement, an entirely mechanical method was used, employing pairs of parallelogram flexure hinges actuated by set screws driven through gear reduction. The STM was also configured such that the sample is mounted on the piezoelectric scanner and the probe is fixed. This allowed the sample to be positioned close to the objective aperture of the SEM. The role of the SEM was envisioned primarily as an alignment tool to position the STM probe over a region of interest on the sample. The STM would then complement the SEM by revealing different surface detail. The instrument was successfully used this way, to image small structures such as Molecular Beam Epitaxy (MBE) and Metal Oxide Chemical Vapor Deposition (MOCVD) grown GaAs/AlGaAs heterostructures and long gate MOSFETs. It was also used to measure the depth of e-beam fabricated calibration pits. Two methods of STM lithography were investigated. Field evaporation of probe material produced mixed results, due in part to the experiments being done in vacuum. Field evaporation proved a useful method for clearing contamination from the probe. STM probe induced modification of passivated silicon surfaces was also investigated. A voltage threshold for depassivation was found and a model for formation of etch masks at positive polarity was proposed. Patterns were written and successfully transferred to the substrate by wet chemical etch. Lines were as thin as 20 nm, and up to 15 nm high. An important result of the lithography research was the discovery that the SEM could image depassivated regions at low accelerating voltages. This allowed some light to be cast on a number of STM imaging artifacts, as well as allowing precise calibration of imaging range. Artifacts included the effect of a finite tip radius on imaging steps and grooves, imaging on highly contaminated surfaces, multiple tips and dielectric material acquired by the probe near the tunneling junction. The effect of electron beam induced carbon deposition was investigated. The STM was used to measure the depth of thin carbon deposits. A deposition rate of between .6 and 2 nm/sec at 300,000X was found.Science, Faculty ofPhysics and Astronomy, Department ofGraduat

Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA.

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues

Assembly of the Complete Sitka Spruce Chloroplast Genome Using 10X Genomics’ GemCode Sequencing Data

<div><p>The linked read sequencing library preparation platform by 10X Genomics produces barcoded sequencing libraries, which are subsequently sequenced using the Illumina short read sequencing technology. In this new approach, long fragments of DNA are partitioned into separate micro-reactions, where the same index sequence is incorporated into each of the sequencing fragment inserts derived from a given long fragment. In this study, we exploited this property by using reads from index sequences associated with a large number of reads, to assemble the chloroplast genome of the Sitka spruce tree (<i>Picea sitchensis)</i>. Here we report on the first Sitka spruce chloroplast genome assembled exclusively from <i>P</i>. <i>sitchensis</i> genomic libraries prepared using the 10X Genomics protocol. We show that the resulting 124,049 base pair long genome shares high sequence similarity with the related white spruce and Norway spruce chloroplast genomes, but diverges substantially from a previously published <i>P</i>. <i>sitchensis- P</i>. <i>thunbergii</i> chimeric genome. The use of reads from high-frequency indices enabled separation of the nuclear genome reads from that of the chloroplast, which resulted in the simplification of the de Bruijn graphs used at the various stages of assembly.</p></div

A high-throughput pipeline for DNA/RNA/small RNA purification from tissue samples for sequencing

High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNA and small RNA, called GSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recovery is presented. GRAB-ALL was benchmarked against existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation

Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

Background RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.Medicine, Faculty ofScience, Faculty ofNon UBCMedical Genetics, Department ofMicrobiology and Immunology, Department ofReviewedFacult