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PacBio Sequel System and Applications

Iso-Seq on PacBio
PacBio Sequel Systems at Novogene

The PacBio Sequel System is the newest SMRT sequencing platform that provides ultra-long sequencing reads with high consensus accuracy, uniform coverage, and simultaneous epigenetic characterization at single-molecule resolution. The PacBio Sequel System provides 7x more throughput, more scalability, and lower sequencing costs than the PacBio RS II System, while maintaining the same benefits. With the addition of the PacBio Sequel Systems that Novogene acquired in early 2017, Novogene now has the world’s largest SMRT sequencing capacity.

The PacBio Sequel System provides unmatched sequencing depths for genomes, transcriptomes and epigenomes. Novogene not only offers PacBio Sequel-based de novo sequencing services, but also provides combined services using various platforms, including Illumina HiSeq, PacBio Sequel, and 10X Genomics Chromium, to match the needs of researchers. Please consult our scientists for the best sequencing strategy to achieve the most comprehensive de novo assembly view for your genome of interest. Novogene’s superior high performance computing and data analysis platform enables high-standard complex genome assembly and fast data analysis.

SMRT Sequencing Advantages

Long average read lengths: average > 10kb, some reads > 60kb
High consensus accuracy: >99.999% (QV50) consensus accuracy
Uniform coverage: no amplification required and even coverage across GC content
Simultaneous epigenetic characterization: directly detect DNA modifications using polymerase kinetics
Single-molecule resolution: Circular Consensus Sequencing (CCS) produces high intra-molecular consensus accuracy

PacBio Sequel System Applications

A. Human Whole Genome Resequencing
B. De novo Sequencing and Assembly of Plant and Animal Genomes
C. De novo Sequencing and Assembly of Microbial Genomes
D. Isoform Sequencing (Iso-Seq) and Full-length Transcripts Generation

A. Human Whole Genome Resequencing

Novogene offers human whole genome resequencing services with PacBio Sequel at multiple coverage levels, providing comprehensive solutions for disease mechanism and studies, cancer research, and haplotype studies. The PacBio Sequel system generates more complete and accurate human whole genome sequences and complements missing sequencing reads, especially in highly polymorphic and highly repetitive regions from short reads sequencing. It also detects novel variations in chromosomal structural variants and fusion genes, and shows the full spectrum of all other variant types, such as copy number variations (CNV), single nucleotide variations (SNV), and insertions / deletions (InDel).

Data Analysis Pipeline:

Figure 1. Bioinformatics analysis pipeline of human whole genome resequencing

B. De novo Sequencing and Assembly of Plant and Animal Genomes

The SMRT sequencing provides uniform coverage and delivers more complete genomes without amplification. The ultra-long reads and uniform coverage also solve the sequencing and assembly difficulties encountered with highly repetitive regions, transposon regions, and highly variable regions, and leads to longer contigs and scaffolds. Thus, the Sequel system is ideal for large genome projects, including human, plant, and animal genomes, as this system can rapidly and cost-effectively generate high-quality de novo assemblies of whole-genome.
Below are some common strategies that we recommend for de novo sequencing of plant and animal genomes based on individual project needs:

Table 1. Sequencing strategies for de novo genome sequencing in plants and animals

Genome Characteristics Assembly Strategy Sequencing Strategy Genome Delivered
Genome with draft reference Gap filling 10x PacBio Sequel reads
Simple genome Hybrid assembly 100X Illumina reads + 20X PacBio Sequel reads + 10X Genomes Contig N50 > 500Kb,
Scaffold N50 > 1Mb
Complex genome Contig N50 > 200Kb,
Scaffold N50 > 1Mb
Simple genome High-quality de novo assembly 70X PacBio Sequel reads Contig N50 > 1Mb
Complex genome Contig N50 > 200Kb
Simple genome Super-scaffold and chromosome scale de novo assembly (Recommended) 80X PacBio Sequel reads + 10X Genomics/BioNano/Chicago/Hi-C Contig N50 > 1Mb
Scaffold N50 > 3Mb
Complex genome Contig N50 > 500Kb
Scaffold N50 > 1Mb

Click here to learn more about de novo sequencing for plant and animals genomes.

C. De novo Sequencing and Assembly of Microbial Genomes

The ultra-long reads from PacBio Sequel System can cover the entire repeat regions of bacterial genomes, providing complete (zero gap) sequences. Assemblies of chromosomal scale also become possible for fungi with this system, and the full chromosomal sequences generated allow individual strains and species to be compared, for example, in evolutionary studies. See below for some recommended strategies.

Table 2. Sequencing strategies for de novo genome sequencing in microbes

Genome Characteristics Assembly Strategy Sequencing Strategy Genome Delivered
Bacteria complete genome map Complete map Over 50X PacBio Sequel reads 1 Scaffold, 0 gap
Fungi fine genome map Genome survey and chromosome-scale assembly 20X Illumina reads + 70X PacBio Sequel reads Contig N50 > 1Mb (simple fungus)
Contig N50 > 500Kb (complex fungus)

Click here to learn more about de novo sequencing for microbial genomes.

D. Isoform Sequencing (Iso-Seq) and Full-length Transcripts Generation

Iso-Seq sequencing, based on the extraordinarily long reads available with the SMRT technology, provides full-length reads spanning entire transcript isoforms from the polyA-tail to the 5′ end without the need for assembly. Iso-Seq is ideal for improving annotations in reference genomes, characterizing gene isoforms, alternative splicing and gene fusion events, as well as complementing the short / incomplete transcripts for species without a reference genome.

Click here to learn more about our Iso-seq service.

Figure 2. Iso-Seq data analysis pipeline

Novogene Project Examples

Journal Title
Nature, 549, 379–383 (2017) The Apostasia genome and the evolution of orchids.
Nature Plants, 3, 946-955 (2017) The Aegilops tauschii genome reveals multiple impacts of transposons.
Nature Communications, 8:1721 (2017) Scallop genome reveals molecular adaptations to semi-sessile life and neurotoxins.