Whole Genome Sequencing on PacBio Sequel II by Nucleome

Nucleome Informatics is the only company in India to offer sequencing services on PacBio Sequel II sequencer. We offer Whole-genome sequencing service of Microbe, Plant and Animal Genomes. 

Sample Requirement

  1. Ten (CLR) to fifteen (CCS) ug of HMW gDNA of more than 50Kb long without any impurity
  2. Low Input DNA library is alos available. Please check with the technical team before placing the order.
  3. You can avail our HMW gDNA prep service too if you wish to send the tissue sample

HMW gDNA and Library QC

  1. Qubit
  2. Bioanalyser
  3. FEMTO Pulse
  4. PFGE

HMW gDNA Shearing and Size selection

  1. Shearing on Megaruptor 3
  2. Size Selection on Blupippin

Library Insert Size

  1. De novo assembly HiFi CCS Library (15 to 20Kb)
  2. De novo assembly CLR Library (30 to 40Kb)
  3. De novo assembly for low DNA input (20Kb)
  4. Microbial de novo assembly CCS Library (10-15Kb)
  5. Variant Detection CCS library (15 – 20Kb)
  6. Structural variant detection CLR library (>15Kb)

Sequencing on Sequel II platform

  1. PacBio Raw Coverage:
    1. Diploid genome: 50 to 60x (CLR) or min 30x-50x Hifi CCS coverage
    2. Polyploid Genome: 30x per haplotype (CCS)
    3. Complex Genome with large repeats: 120X coverage (CCS or CLR)
    4. Microbial Genomes: 200x CCS HiFi Coverage to achieve at least 30x Unique Molecule coverage per microbial genome
    5. Variant Detection: More than 15X HiFi for a human genome
    6. Structural Variant Detection: 5 to 25x CLR coverage

Data analysis

We use various tools to assemble the genomes. We build contig level assembly with PacBio Sequel II data and scaffold the genome with Optical map and HiC data after multiple rounds of polishing, gap filling and curation. Assessment of assembly is done using assembly stats like Contig N50, Scaffold N50, No of Gaps, Base accuracy, BUSCO Score, RNA Seq mappability, Phase Block resolution and Chromosomal level continuity. We offer the following type of assembly and scaffolding services;

  1. De novo contig level mosaic assembly
  2. Haplotype Phased diploid genome Assembly
  3. Trio Binning assembly
  4. Microbial genome assembly
  5. Polyploid genome assembly
  6. Scaffolding with Optical mapping 
  7. Superscaffolding with HiC data
  8. Polishing and error correction
  9. Genome Quality Assessment 
  10. Assembly Curation of previously developed genome assembly

For any query and more information on our services, please contact our sales team at salessupport @ nucleomeinfo.com. 

Pacific Biosystems PacBio Sequel II product.
20190322_10290_PacBio_Sequel2.CR2
Megaruptor 3 at Nucleome

ISO Sequencing

Nucleome provides full-length transcriptome sequencing i.e ISO Sequencing on Pacbio Sequel II platform. Nucleome Informatics is the only service provider of ISO Sequencing on Sequel II in India. The PacBio Iso-Seq method is an end-to-end workflow for sequencing and analyzing full-length transcript isoforms. Our Iso-Seq analysis enables you to detect novel transcripts and genes, identify fusion genes, annotate isoforms and alternative splicing events.

Generate full-length transcript sequences up to ~15kb

  • High accuracy (>99%) for ORF prediction
  • No reference genome required
  • Bioinformatics tools from raw data to functional annotation

ISO Seq Service WorkFLow:

1. Convert RNA into cDNA

2. cDNA SMRTbell library preparation

3. Sequence on the Sequel II System

4. Generate circular consensus sequences (CCS) 

5. Discover isoforms de novo with Iso-Seq analysis

ISO Seq Applications:

Whole-genome Annotation

-Typically whole-transcriptome, non- quantitative

-Often included in de novo genome assembly projects

-Single tissue to several tissues

-Generates reference transcriptome for downstream RNA-seq studies

Gene-level Isoform Discovery

-Typically targeted, either cDNA amplicons or target capture

-Useful for detecting gene fusions, SNVs, allele-specific expression

-Cost-effectively multiplex many samples per single SMRT Cell

-Relative quantitation possible

Sample Requirement

1 ug of high qulaity total RNA, RIN >7 and Concentration should be more than 25ng per ul

– Has not been exposed to high temperatures (e.g.: > 65°C for 1 hour can cause a detectable decrease in sequence quality) or pH extremes (< 6 or > 9).

– Has an OD260/OD280 ratio between 2.0 and 2.2. – Has an OD260/OD230 ratio between 1.8 and 2.1. – Has a RIN number ≥ 8 (Recommended).

– Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation. SYBR dyes are not RNA damaging, but do avoid ethidium bromide.

– Does not contain denaturants (e.g., guanidinium salts or phenol) or detergents (e.g., SDS or Triton‐X100).

– Does not contain carryover contamination from the original organism/tissue (e.g., heme, humic acid, polyphenols, etc.).

– Note: RNA samples should only be shipped with dry ice.

Contact our team now to receive the quote and place the order.

Nucleome is the only service provider in South Asia that offers Single Molecule, Real-Time (SMRT) Sequencing on PacBio Sequel II that generates the long, accurate, single-molecule reads you need to comprehensively characterize samples with complex variation. With SMRT Sequencing you can:

  • Identify closely related individuals within a heterogeneous mixture
  • Track the rapid evolution in response to environmental conditions, immune pressures, or drug treatments

Highly accurate long reads – HiFi reads – with the single-molecule resolution are ideal for full-length 16S rRNA sequencing, shotgun metagenomic profiling, and metagenome assembly so that you can:

  • Determine community composition at the species or strain-level with competitively priced full-length16S sequencing
  • Identify 6-8 full-length genes in every HiFi read with efficient, cost-effective metagenomic profiling
  • Generate up to 20 high-quality metagenome-assembled genomes (MAGs) per multiplexed human faecal sample with just 6 Gb of HiFi data
  • Leverage epigenomic data to associate contigs and plasmids from closely related strains

Full-length  Metagenome assembly and functional profiling on PacBio Sequel II

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly.

HiFi sequencing can be used even for sample types with limited starting material, allowing for reference quality MAGs, discovery of novel genes without the need for de novo assembly, identification of biochemical pathways, and resolution of taxa below the species level with confidence.

  • Unbiased compositional and functional characterization of microbial communities
  •  Generation of complete or near- complete genome assemblies from microbial populations

Data analysis:

Output data in standard file formats, (BAM and FASTA/Q) for seamless integration with downstream analysis tools – Perform taxonomic classification and functional gene profiling using QIIME and MEGAN
– Perform gene prediction and discovery using FragGeneScan and Prodigal

Learn more about workflows for these types of complex populations

– Perform metagenomic shotgun assembly directly with HiFi reads using HiCanu

Full length 16s Amplicon Sequencing by Nucleome

Nucleome is the only service provider in South Asia that offers Single Molecule, Real-Time (SMRT) Sequencing on PacBio Sequel II that generates full length 16s amplicon sequencing. The ~1500 bp 16S rRNA gene comprises nine variable regions interspersed throughout the highly conserved 16S sequence. Sequencing the partial gene was originally accomplished by Sanger sequencing. This required cloning genes, generating, and assembling two to three reads per clone, and producing limited sampling depth at high cost and effort. The partial 16s rRNA gene sequencing misses to sequence the entire taxa of the bacterial community.

DIFFERENT SUB-REGIONS SHOWED BIAS IN THE BACTERIAL TAXA THEY WERE ABLE TO IDENTIFY

  • V4: Consistently poor performance
  • V1–V2: poor for Proteobacteria
  • V3–V5: poor for Actinobacteria
  • V1–V3: good results for Escherichia / Shigella
  • V3–V5: good results for Klebsiella, 
  • V6–V9: good results for Clostridium and Staphylococcus 

Full V1–V9 region: the only way to resolve ALL the clades that may be present in a complex population. Order full length 16s Amplicon sequencing to Nucleome and discover Strain-level identification of bacterial communities with the unprecedented accuracy of PacBio HiFi reads.