De Novo Genome Sequencing

Nucleome is India’s only service provider of Whole-genome sequencing and hybrid assembly services using Illumina NovaSeq 6000 and PacBio Sequel II. To sequence a genome of an organism, we use PacBio Sequel II, optical mapping and HiC approaches. We have completed genome sequencing & assembly of many organisms like microbes, fungus, plants, insects and animals using Illumina NovaSeq 6000 and PacBio Sequel II. Depending on the sample type and budget available, we can design a suitable strategy for your project. Visit PacBio Sequel II service page for more information. If you wish to sequence a vertebrate genome, we are the most trained team in India. In Partnership with the Vertebrate Genomes Project, Nucleome is working with Indian researchers and VGP to sequence 100 vertebrate genomes from India. 

For very high-quality genome assembly, we use Pacbio Sequel II data, scaffold with Bionano datasets and finally use HiC to build chromosome level scaffolds. We perform the assembly assessment and curation multiple times to ensure scientists get the error-free scaffolds from available data sets. We were involved in sequencing genomes of Tiger, Great Indian Bustard, Mango, Pigeon Pea, Pomegranate, Mithun and many microbial and fungal genomes. 

 

Our team can assist you in designing the project, selection of technology, sequencing and analysis. Call our team now at +91 40 4011 4169 or send a query to info @ nucleomeinfo.com.

Optical Mapping

Optical mapping is a molecular technique that produces fingerprints of DNA sequences in order to construct genome-wide maps. The sequence markers can be ordered restriction fragments, or specific sequence motifs (nick sites). The optical mapping procedure first stretches relatively intact (minimally-sheared) linear DNA fragments on a glass surface or in a nanochannel array, and then directly images the locations of the restriction sites or sequence motifs under light microscopes, with the aid of dye or fluorescent label. Optical mapping has been widely used to improve de novo plant genome assemblies, including rice, maize, Medicago, Amborella, tomato and wheat, with more genomes in the pipeline. We use Bionano Irys system to offer optical mapping service that provides long-range information of the genome and can more easily identify large structural variations. The ability of optical mapping to assay long single DNA molecules nicely complements short-read sequencing which is more suitable for the identification of small and short-range variants.

Optical Map guided genome assembly

There are several ways in the assembly process that optical mapping can assist in building high quality reference genomes. De novo constructed optical maps offer independent evidence to connect and bridge adjacent sequence contigs or scaffolds. Genome assemblies guided by optical maps consist of three key computational steps. The initial step is the de novo assembly of optically mapped molecules to construct a ‘consensus’ optical map from single DNA molecules at high redundancy. The consensus map has to deal with errors specific to optical mapping including missing cuts, false cuts, inaccurate fragment sizes, and chimeric maps. The next step is to align the in silico digested contig sequences to the consensus optical map. The final step is the joining of neighbouring contig sequences to construct supercontigs on the basis of their locations on the optical map. For small microbial genomes, the resulting assemblies could contain a single extent of sequence that spans the entire genome, while for large eukaryotic genomes the combined efforts of sequencing and optical mapping often result in substantially increased scaffold N50. In several cases, the mapping data allow the reconstruction of entire chromosomes. Beyond ordering and orientating contigs, optical maps provide an additional layer of validation to the sequence assemblies. Optical maps could potentially identify and resolve misassemblies – false joins, inversions or translocations that are artifacts, which occurred during the sequence assembly.

Genome Resequencing

Whole genome re-sequencing approach can be used to the underlying mechanisms of species origin, development, growth, and evolution. Using Whole Genome Resequencing, the complete genome data from one or more variants can be aligned to known reference genome of the species. Applications of WGS include detection of genetic differences between variants, transposon fingerprinting for assessing germplasm diversity and lineages, and mapping loci associated with specific traits, such as disease resistance.

 

We prefer Illumina sequencers for genome resequencing. PCR free libraries and coverage of minimum 30x is recommended for better results.

Human Genome Sequencing

Nucleome offers highly precise, inclusive human whole-genome sequencing services, giving researchers, Physicians and patients the clearest picture of the genome. During whole genome sequencing, we collect a DNA sample and then determine the identity of the 3 billion nucleotides that compose the human genome. Today, most genetic testing focuses on one or a few genes, rather than the entire genome. However, with the availability of human genome sequencing service in India at Nucleome Informatics, now more individuals are pursuing this option. Physicians can look at an entire genome to see how specific treatments for a disease will be affected by an individual’s unique genome. For example, the physician may opt to look at genes involved in drug metabolism when deciding dosage. In the future, whole genome sequencing may enable everyone to develop a personalized treatment plan.

Advantages of Whole Genome Sequencing

  • Creating personalized plans to treat disease may be possible based not only on the mutant genes causing a disease, but also other genes in the patient’s genome.
  • Genotyping cancer cells and understanding what genes are misregulated allows physicians to select the best chemotherapy and potentially expose the patient to less toxic treatment since the therapy is tailored.
  • Previously unknown genes may be identified as contributing to a disease state. Traditional genetic testing looks only at the common “troublemaker” genes.
  • Lifestyle or environmental changes that can mediate the effects of genetic predisposition may be identified and then moderated.

Sequencing Strategy

  • 350 bp insertion DNA library.
  • Illumina HiSeq Platform, Paired-end150 bp.
  • Sample Requirements.
    • DNA amount quantified by Qubit 3.0
    • For fresh sample: ≥2.0 μg (for two libraries prep); minimum: 500 ng
    • For FFPE sample: ≥3.0 μg (for two libraries prep); minimum:1μg
    • DNA concentration: ≥20 ng/μL
    • Total volume: ≥10 μL
    • Purity: OD260/280= 1.8-2.0 without degradation or RNA contamination
    • Turnaround Time: Within 45 days from sample verification Additional 15 days for standard bioinformatics analysis
  • Recommended Sequencing Depth
    • For normal sample: effective sequencing depth 30X
    • For tumor sample: effective sequencing depth 50X

Bioinformatic Analysis

  • Data quality control: filtering reads containing adapter or with low quality
  • Alignment with reference genome, statistics of sequencing depth and coverage
  • SNP/InDel/SV/CNV calling, annotation and statistics
  • Somatic SNP/InDel/SV/CNV calling, annotation and statistics (paired tumor samples)

For more information please visit our Youtube Channel;

If you are interested to sequence your genome and need more information, please write to us at hgs@nucleomeinfo.com.

Exome Sequencing

Nucleome offers Exome sequencing, a cost-effective approach to whole genome sequencing as it targets only the protein coding region of the human genome responsible for a majority of known disease related variants. Whether you are conducting studies in rare Mendelian disorders, complex disease, cancer research, or human population studies, Nucleome’s comprehensive  whole exome sequencing service provides a high-quality, affordable and convenient solution. We use Agilent SureSelect Human All Exon V5/V6 Kit for exome capture and Illumina HiSeq 2500 for sequencing. We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, which exceeds Illumina’s official guarantee of ≥ 75%.

 

We offer bioinformatics analysis too that includes data QC, mapping with reference genome, SNP/InDel, somatic SNP/InDel calling, statistics and annotation. Nucleome utilizes internationally recognized software in bioinformatics analysis, e.g. BWA, SAMtools, GATK, etc.

Single Cell DNA Sequencing

The genomic heterogeneity of cell populations can be explored at the level of the individual cell. Genetic changes, such as point mutations and copy number variation occurring during disease and normal development processes, are profiled using the minute amounts of DNA from single cells. Applications include analysis of genetic heterogeneity within unicellular and multicellular organisms, detection of chromosomal anomalies in germ line cells, preimplantation genomic screening of embryos, and defining the genetic composition of tumors for developing more targeted therapies.

 

The single cell DNA sequencing service includes sample QC, amplification, library preparation, sequencing and bioinformatics analysis. We use the MALBAC (multiple annealing and looping based amplification cycles) PCR-based method, which provides uniform data while reducing rates of false positives and false negatives.

Genotyping By Sequencing

This application is used to compare genotypes through the mapping of large numbers of SNPs or other markers. Genotyping by sequencing (GBS) is a rapid and cost-effective approach which uses a restriction enzyme digestion step to reduce genome complexity, so GBS can be applied to large genomes, and the end reads of the restriction fragments allow variants to be compared when no reference genome is available.

 

Applications of genotyping by sequencing include tracking plant and animal genotypes in breeding programs and conservation projects, examining the diversity of natural populations, discovery of new genetic markers, and screening variants prior to whole genome re-sequencing.