Pyrosequencing (Roche) – sequencing by synthesis, detects light when pyrophosphate is released.
Sequencing by synthesis (Ilumina) – detects colour.
Single molecule sequencing by synthesis (Heliscope)
Sequencing by ligation (ABI)
NextSeq 500 – 400 million reads of 500bp.
Nanostring technology – target RNA, probe with 7 fluorescent RNA segments. The segments are in different orders for each probe, and therefore each gene. Shows gene expression. Need capture probe and reporter probe. Quantify and sequence. One fluorescent molecule = one count in digital analyser. Used for cancer patients to give risk of getting ill again. 3D – DNA, RNA and protein.
Digital drop PCR – A sample is fractionated into 20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet.
Nanodrop – DNA, RNA and protein quantitation with only 1–2 µL of sample
Electrophoresis – migration and separation of charged particles (ions) under the influence of an electric field.
Microarray – agilent vs affymetrix
Started with sanger, then became capillary to see SNPs
Ion torrent – detect electric change
Illumina – detect colour
Applications of NGS – Whole genome sequencing, exome sequencing, RNA seq, methylation seq.
TruSeq – 1.2ug, PCR free, avoids PCR amplification errors, 350 or 550 bp, validate using qPCR.
TruSeq Nano – 100-200ng, small amount of DNA, amplification, validate using qbit.
Nextera – for large complex genomes, shorter prep time. Tagmentation – cuts and adds adapter at same time. Qbit and bioanalyser. Eg human genome.
Nextera DNA XT – small genomes, eg bacteria, no quantification.
Exome Seq – cheaper than WGS. Around 2% of genome.
Exons and introns and regulatory regions
Truseq total RNA
Truseq stranded mRNA
Truseq small RNA and small like miRNA – no depletion or enrichment. RT PCR. Run on gel. 147 + 157bp. Bioanalyser.
Ethical issues – do you want to know future? Insurance?