NGS Analysis

Next-Gen Sequencing

Next-Generation Sequencing (NGS) involves high-throughput sequencing technologies that have, over time, become affordable to most labs. The ability to generate large volumes of data has greatly empowered genetic, biological and medical research.

Sequencing technologies involve three main processes in the following order: library preparation, sequencing and analysis. The choice of technology or combination of technologies depends on the purpose of the project; for example, resequencing vs de novo sequencing and assembly will require different approaches.

AgriGenome offers a comprehensive range of next-generation sequencing services utilizing multiple sequencing platforms including Illumina HiSeq, MiSeq, PacBio Sequel and Oxford Nanopore Technology. Our team of scientists and Bioinformaticians work with you to deliver reliable high-quality results and provide end-to-end solutions for your NGS project needs.

Exome & Targeted Resequencing

Exome sequencing (also known as targeted exome capture) is a strategy to selectively sequence the coding regions of the genome and is a more affordable alternative to whole genome sequencing. In the human genome, there are about 180,000 exons: they make up about 1% of the human genome, which translates to about 30 megabases (Mb) in length.
It is estimated that the protein coding regions of the human genome account for about 85% of the disease-causing mutations. Exome sequencing is a productive and cost effective method to identify functional variations that are responsible for both Mendelian and common diseases.
Enrichment methods available with us:

• Illumina TruSeq Exome Enrichment

• Nimblegen Sequence Capture E

• Agilent SureSelect Human All Exon

• Additionally, Custom enrichment is also available

De Novo Genome Sequencing

De Novo genome sequencing is used to characterize genomes where, either there are no reference sequences available, or in known genomes to study structural variations. De Novo sequence reads are preferably generated on those NGS platforms that generate longer sequence reads like Oxford Nanopore Technologies or PacBio SMRT; De Novo sequencing can also be done on short read technologies like Illumina HiSeq platforms with different insert sizes (Paired end and mate pair).

Sequence reads from multiple platforms can be combined for Hybrid assembly (e.g., Illumina + PacBio Sequel) for cost effective coverage of large genomes. Sanger sequencing reads can also be used to complete the coverage.

AgriGenome offers Illumina HiSeq, MiSeq, Oxford Nanopore Technologies, PacBio Sequel and Sanger services for De Novo genome sequencing and also Bioinformatics data analysis for efficient assembly, gene prediction and annotation.

Transcriptome (RNA-Seq)

RNA-Seq is a powerful approach that enables researchers to discover, profile and quantify RNA transcripts in the whole transcriptome. As this method does not use predesigned probes or primers, it becomes an unbiased, hypothesis-free approach providing researchers with applications that are not possible by traditional microarray-based methods.

RNA sequencing is used widely for:

• Studies of gene expression and discovery of novel transcripts and isoforms

• SNP discovery in the coding portions of the genome

• De Novo transcriptome assembly

The basis for expression quantification in RNA-Seq

  1. Counting or Profiling
    10 million total reads from poly-A selected RNA will give performance better than any microarray
  2. Studying Alternative Splicing or quantifying cSNPs for most transcripts
    Deeper profiling of 50 to 100 million reads, with reading lengths of 50 to 100bps, from poly-A selected RNA, will enable studies on alternative splicing and quantification of cSNPs for most transcripts.
  3. Complete Annotation of an entirely New Transcriptome
    ~500 Million reads of 100 bp read length from multiple tissues
    Platform: We offer Transcriptome sequencing on the Illumina Hiseq platform

Metagenomics:-

Metagenomics, also known as Environmental genomics or Community genomics, is the study of multiple microbial species present in a specific sample that usually harbours complex microbial communities. Using NGS, it is possible to study microbial communities by sequencing the gene of choice (e.g. 16S, V3 and/or V4 regions) or the whole genome or transcriptome without the need for culturing.
Platform: Metagenomic sequence reads are generated on Illumina MiSeq/ HiSeq or PacBio Sequel depending on the experimental objectives. We can do a detailed analysis of the metagenome NGS data.

Small RNA Sequencing:-

Small RNA sequencing is a powerful application enabling the discovery and profiling of microRNAs and other non-coding RNAs in any organism, without prior genome annotation. Using low RNA inputs, you can profile the differential expression of known microRNAs as well as detect novel microRNA targets and wide-ranging sequence variation or “iso-miRs” present in miRBase accessions.

With unprecedented sensitivity and dynamic range, small RNA sequencing methods allow for the most accurate detection and quantification of rare small RNA sequences.

Platform : We offer Transcriptome sequencing on Illumina HiSeq platform

ChIP-seq (Chromatin Immunoprecipitation-Sequencing)

ChIP-seq is a technique or methodology to map transcription factor binding sites and histone modification status on a genome-wide scale. ChIP (Chromatin immunoprecipitation) technique comprises a few basic steps: cross-linking a protein to chromatin, shearing the chromatin, using a specific antibody to precipitate the protein of interest with its associated DNA, and reversing the cross-linking and finally purifying the associated DNA fragments.

Once ChIP DNA (200-800bp in size) is submitted for sequencing, ChIP DNA libraries are made and run on Illumina MiSeq or HiSeq. This often generates tens to hundreds of millions of 50-100 bp sequences (also called short reads) from the 5′ ends of ChIP-DNA fragments. We also do advanced data analysis of ChIP-seq data.

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