CCR Sequencing Facility

Established Technologies

We have two NovaSeq XPlus, two NovaSeq 6000, two Nextseq 2000,   one MiSeq, one 10X Genomics, Mission Bio, PIP seq,   PacBio Revio and Sequel, Oxford nanopore (ONT) technologies for our sequencing and Optical Mapping Bionano.

De Novo Sequencing

• Paired-end reads – Generate long scaffolds and contigs using multiple insert lengths
• Read length – Use paired-end reads in excess of 300 base pairs for mammalian-scale de novo assembly

Resequencing

• SNP discovery and confirmation
• Insertions and deletions (indels) and copy number variations (CNVs)
• Structural variant discovery

Transcript Profiling and Discovery

• Characterize splice variants, coding SNPs, and relative expression of transcripts.
• mRNA-Seq, tag profiling, and small RNA analysis
• Measurement of alternative isoforms, discovery of novel structures and coding SNPs

Bisulfite Sequencing and DNA-Protein Interactions

• Detection of variations in methylation signatures at single-base resolution
• Sequencing of repetitive bisulfite-converted genomes using proprietary reversible-terminator chemistry
• CpG methylation, histone modifications, chromatin structure, or DNA-protein interactions
• Chromatin immunoprecipitation-paired sequencing approach (ChIP-Seq)

PacBio Sequel Sequencing

• Sequencing via single molecule, real-time (SMRT) technology allows rapid identification of long nucleotide chains.
• Read lengths averaging greater than 6,000 bases per molecule, with maximum read lengths > 30,000 bases, facilitate genome assembly and mapping of repetitive regions.
• Amplification-free direct sequencing of individual molecules precludes PCR bias and artifacts.
• Low-input Protocol developed by the Sequencing Facility enables sequencing of as little as 500 picograms of starting material for many applications.
• Minimal machine turnaround time provides flexibility in experimental and run design.

Developing Technologies

Nabsys: High-Definition Mapping (HDM) using electronic detection of tagged single high molecular weight (HMW) DNA molecules

• HDM provides routine, accurate, cost-effective analysis of genomic structural information, unavailable with short read technologies.

• These characteristics make HDM an ideal first-line approach for a variety of applications for small and large genomes, including de novo map assembly, structural variant analysis, hybrid assembly, metagenome characterization and strain identification.

Xdrop-Sort: Target DNA enrichment for SV or virus integration detection

• A novel microfluidic-based system that allows for targeted enrichment of long DNA molecules using only a few nanograms of DNA.

• Based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted. The final product is an enriched population of DNA molecules that can be investigated by long read sequencing.

• Single cell RNA-seq applications will be coming soon with the release of a new cartridge from Samplix.

New Applications:

Cell-free DNA sequencing: Cell-free DNA (cfDNA) refers to all non-encapsulated DNA in the bloodstream. cfDNA are nucleic acid fragments that enter the bloodstream during apoptosis or necrosis. A portion of that cell-free DNA may originate from a tumor clone and is called circulating tumor DNA (ctDNA). cfDNA sequencing will therefore provide a quick and easy way for early cancer detection. The R&D team is currently in the process of developing adapted protocols for short read and long read cfDNA sequencing.

Single cell RNA-seq on ONT with or without adaptive sampling depletion/enrichment: Single cell RNA sequencing (scRNA-seq) technology has become the state-of-the-art approach for unraveling the heterogeneity and complexity of RNA transcripts within individual cells, as well as revealing the composition of different cell types and functions within highly organized tissues/organs/organisms. ONT’s high-throughput long read sequencer, PromethION, can sequence full length cDNA generated from single cell RNA-seq captures and detect not only the gene expression, but also the isoform information at single cell level. The adaptive sampling on ONT can selectively sequence the interested genes and increase the coverage of the region of interest. Our R&D team is establishing the protocol for single cell RNA Iso-seq on ONT with or without adaptive sampling.

ResolveOME single-cell whole genome and transcriptome amplification from BioSkryb genomics: BioSkryb Genomics has developed a unified system named ResolveOME for single cell whole transcriptome and whole genome amplification sequencing analysis. The ResolveOME system allows comprehensive analysis of the transcriptome and genome in parallel from the same cell. It provides high resolution accuracy of genome analysis down to the single base level combined with the comprehensive full length mRNA transcriptome and enables the understanding of interplay of these omic layers within and between individual cells. Our R&D team is evaluating the performance of this protocol.

Illumina Complete Long Read Sequencing Technology generates contiguous long-read sequences with N50 of 5–7 kb with some reads > 10 kb. It has the potential to improve the efficiency and accuracy of some existing DNA sequencing applications while increasing the resolution of clinically important genes. The technology simplifies de novo sequencing because large repeat regions in the DNA fragments can easily be spanned.

5-hmC and 5mC Detection and Analysis: Discrimination between 5-mC and 5-hmC in CCGG sequences using enzymatic digestion and PCR amplification using the The EpiMark® 5-hmC and 5-mC Analysis Kit. This can also be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a specific locus.