Usually, mate pair reads are combined with pair-end reads to generate deep coverage and accurate mapping. Such long-insert mate pair libraries are useful for a number of sequencing applications, including:
Details on Illumina sequencing library preparation protocols can be found in the genomics datasheet document at this link.
Applications of Illumina Sequencing
Illumina sequencing provides a wide range of tools to probe any given genome, transcriptome or epigenome. Most applications of illumina sequencing methods differ in the ways of gDNA or RNA extraction and library preparation methods while the process of sequencing remains the same. There are a number of library preparation kits available from Illumina and other vendors for a specific application. At 1010genome we have optimized sample extraction methods based on a specific application and organism type to deliver high quality illumina sequencing reads.
These are the key applications areas for Illumina Sequencing:
Whole Genome Sequencing
Rapid drop in Illumina sequencing has made it easier to probe every base in a given genome to identify SNPs, Indels and SVs. Human, livestock, plant and diseases causing bacterial genomes are being sequenced rapidly to understand diseases, improve longevity, identity industrially important genes and pathological pathways.
1010genome provide high quality whole genome sequencing and bioinformatics analysis services to identify SNPs, indels, SV and biomarkers for whole genome studies. Details of whole genome sequencing and our services are available here.
Whole Exome Sequencing
Exomes harbor 85% of medelinian diseases causing SNPs thus making exome sequencing a key application. Researchers have a choice to perform exome enrichment or to sequencing full genomes to later focus on exomes. With a ever dropping price of illumina sequencing, researchers are now choosing to sequence whole genomes instead of enrichment methods.
1010genome provide high quality exome genome sequencing and bioinformatics analysis services to identify SNPs, indels, SV and biomarkers for exome studies. Details of whole exome sequencing and our services are available here.
De novo Assembly
In absence of a reference sequence for novel species, sequencing reads can be assembled into contigs that provides a map of genome. Illumina sequencing provides deep coverage at affordable costs making it easier to perform de novo assemblies for genome. Mate pair libraries help to enhance the N50 size and contiguity of genome drafts. A large number of assembly software are available for de novo assembly. More recently, Pacbio and Oxford Nanopore long read sequencing are also being used for assemblies as these reads are 10kb or longer on average. Each of these next-generation sequencing technology have their advantages and shortcomings for assembly applications.
Refer to the de novo assembly details page for details of assembly services we provide.
This technique allows to study a loci or region of interest at a lower cost and shot time expense. Targeted sequencing allows to generate deep sequencing coverage that helps to identify SNPs and INDELS with high confidence. The two most popular examples of targeted sequencing are exome enrichment and 16s rRNA amplicon sequencing. Details for each of these techniques can be found at:
RNA seq focuses on sequencing mRNA, small RNA, non-coding RNA and micro-RNAs.
mRNA sequencing is the most popular transcriptomics application that provides a great tool to understand differential gene expression across conditions like stress, disease or change of environment across samples.
For details of RNA Seq for gene expression studies please refer to link.
Just like de novo assembly, in absence of a transcriptome draft that provides sequence profile for all mRNA present in an organism, de novo assembly of mRNA reads can be performed. Transcriptome assembly provides an estimate of number of genes, genes structure, alternative splicing, isoform diversity and gene fusion events. Illumina sequencing reads have been popular with researchers to generate a draft transcript assembly that is used as base for gene expression studies. We have developed and optimized transcriptome sequencing protocols and transcript assembly pipelines. For more details please refer to Transcriptomics and assembly page.
Small RNA and Noncoding RNA sequencing
Small, noncoding and micro RNAs are short 18-22 bp long RNA that play a crucial role in regulation of gene expression as they often act as gene silencer or repressor. Illumina sequencing provides a deep coverage to match these against known databases for profilign or identifying novel RNAs. Get in touch with us for consultation on you project.
Epigenomics is the study of heritable changes in gene activity caused by non-DNA changes mainly through DNA methylation, DNA-protein interaction and histone modifications. Role of DNA methylation or cytosine to 5’ methyl-cytosine in gene activity regulation is very well established. Illumina sequencing provides two ways to probe these methylation patterns in a genome – Whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing.
In WGBS, sodium bisulfite chemistry converts non-methylated cytosines (C) to uracils (U), these uracil (U) bases get converted to thymine (T) during sequencing.
In RRBS, DNA is digested with MspI, a restriction enzyme unaffected by methylation status. Fragments in the 100–150 bp size range are isolated to enrich for CpG and promotor containing DNA regions