Long-read sequencing

The KMSR offers long-read sequencing solutions from Oxford Nanopore and PacBio. Compared to short-read sequencing technology, they provide the following advantages:

• Long reads with no amplification.
• Direct detection of epigenetic modifications on native DNA.
• Direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms.
• Uniform coverage of the genome as they are not as sensitive to GC content as short-read platforms.

Oxford Nanopore Technologies Long Read Sequencing System

Oxford Nanopore is a long-read sequencing technology that utilizes an ionic current through nanopores and measures the changes in current as biological molecules pass through the nanopore or near it. The information about the change in current can be used to identify that molecule. Currently YCGA supports one minION device and projects are typically customized to investigator’s specific needs, please contact Guilin Wang to discuss beginning a Nanopore project.

Applications

• Long- or ultra-long read DNA sequencing: Due to the utilization of nanopores, read length is limited by the integrity of the DNA sample, with reads reported as large as 2.3Mb. Two library protocols exist for this application, one utilizes ligase to anneal the nanopore adapters resulting in libraries suitable for long-reads and the second adds adaptors by a transposase. This second method is utilized for the longest possible read lengths.
• Epigenetics: Amplification is not a requirement for Nanopore library preparation. This both facilitates  long-read sequencing and preserves epigenetic features.
• Targeted sequencing: These protocols use the CRISPR/Cas9 system for PCR-free enrichment of highly repetitive or long gene targets. This offers investigators the ability to focus their long-read sequencing without compromises associated with sequencing-by-synthesis.
• Full-length cDNA & direct RNA sequencing: Full-length cDNA sequencing is recommended by Nanopore for those investigators who are interested in splice variants and fusion transcript analysis; with only a reverse-transcriptase step the material is minimally modified. For investigators that wish to directly sequence RNA without RT or PCR bias, RNA can be sequenced directly to preserve qualities such as modified bases. 

PacBio SMRT sequencing system

The Sequel II System provides the advantages of single-molecule real-time (SMRT) sequencing and now makes it more affordable for all scientists to drive discovery with comprehensive views of genomes and transcriptomes.

What are HiFi reads?

HiFi reads are produced using circular consensus sequencing (CCS) mode on PacBio long-read systems. HiFi reads provide base-level resolution with 99.9% single-molecule read accuracy.

HiFi reads can be used across a wide range of SMRT sequencing applications, from whole genome sequencing for de novo assembly, comprehensive variant detection, epigenetic characterization, RNA sequencing, and more.

How are HiFi reads generated?

Provides access to even more highly accurate long reads. Operates in two modes:

• Circular consensus sequencing (CCS) mode to provide base-level resolution with >99% single-molecule read accuracy for the detection of all variant types from single nucleotide to structural variants.
• Continuous long read (CLR) sequencing mode to enable high-quality assembly of even the most complex genomes. Expect half the data in reads >50 kb and the longest reads up to 175 kb.

Applications

• Whole genome sequencing: De novo genome assembly
  • Single molecule real-time (SMRT) sequencing
• Targeted sequencing: No Amp targeted sequencing utilizes a CRISPR/Cas9 system
  • Eliminate PCR bias and errors
  • Sequence through entire repeat expansions with base-level resolution
  • Quantify repeat numbers in normal- and mutant-expanded alleles
  • Identify interruption sequences
  • Characterize somatic mosaicism
• RNA sequencing
  • Discover new genes, transcripts and alternative splicing events
  • Improve genome annotation to identify gene structure, regulatory elements, and coding regions
  • Increase the accuracy of RNA-seq quantification with isoform-level resolution
• Epigenetics
  • Single molecule real-time (SMRT) sequencing directly detects epigenetic modifications by measuring kinetic variation during base incorporation.
  • By capturing these modifications simultaneously with sequence data, this method eliminates the need for special sample preparation and additional sequencing.