Deep Sequencing-Based Applications
Massively parallel sequencing techniques determine the four base sequence of thousands to millions of molecules of nucleic acid in a single assay in short segments of up to 400 nt which can then be aligned together or to a reference sequence. The technique can be applied without the need of prior information on genes or regions to be covered, or targeting specific regions (by specific PCR amplification or by enrichment hybridization capture).
Sequencing based applications can allow both qualitative (base sequence determination) or quantitative analysis (sequence counting). Sequence reads may be assembled de novo or mapped to a reference sequence allowing to determine sequence variation relative to the reference. Read mapping to specific regions or genes and counting allows to perform quantitative profiling or peak calling. Numbers of reads may go from 100.000 (Roche) to up to 1.500.000.000 reads per run (Illumina), with Ion Torrent offering 100.000 to 6.000.000 reads per run. Read lengths may span from short reads from 18 up to 100 nt (Illumina) to up to 400 nt (Ion Torrent) and up to long 800 nt reads (Roche). There is the possibility to bar code and multiplex from 2 to 96 samples per run. There is a minimum number of samples to fill a run which can vary depending on application, reading mode and desired depth of coverage.
DNA-seq (including whole genome, exome sequencing, CHIP-seq, bisulphite sequencing, metagenomic profiling and other applications)
It involves deep sequencing of fragmented adaptor-ligated DNA libraries from either single ends (single read) or both ends (paired end) or joined ends (mate pairs). Starting material may be genomic DNA (individual or pooled from metagenomic samples), immunoprecipitated chromatin or large amplicons and may have been selected through enrichment using targeted hybridization capture. Uses include 1) de novo genome sequencing; 2) resequencing to look for sequence variants (SNPs, small insertions and deletions), copy number alterations or translocations -not detectable by microarrays-; 3) chromatin analysis looking for sites of binding by proteins such as histones of transcription factors or DNA modification by methylation, and 4) species identification in mixed populations (metagenomic profiling). Equivalent to Sanger DNA sequencing but on a massive scale, it is complementary to microarray applications such as SNP genotyping, aCGH, copy number analysis, LOH analysis, CHIP on chip, MeDIP on chip. Please inquire for assay specific details.
RNA-seq (including mRNA-seq, small RNA-seq and other applications)
It involves deep sequencing of fragmented adaptor ligated cDNA libraries with the option of strand specific or non-strand specific starting form total RNA, riboRNA reduced, polyA+ RNA or small RNA allows to profile expression levels, and to determine isoforms derived from alternative promoter, splicing, polyadenylation or transcript fusion in coding and non-coding RNAs. Equivalent to Sanger sequencing of ESTs but on a massive scale, it is complementary to microarray expression profiling. Please inquire for assay specific details.
Amplicon sequencing (including mutation profiling, 16S rDNA sequencing and other applications)
Equivalent to Sanger sequencing of PCR products, It involves, deep sequencing of PCR products generated with fusion primers may be used to characterize genetic variation and detect mutations in defined segments of the genome, or to profile mixed populations of cells by looking at ultra-conserved regions (like 16S ribosomal RNA) for metagenomic profiling. Please inquire for assay specific details.