sequencing by synthesis – almost all technology based on this method. add nucleotide one at a time, attached to stop codon. attaches, see colour, remove stop codon, continue.
ion torrent – sequence by electrical signal. chip. lots of wells that detect an electrical change. add each time a different nucleotide and then wash. if attaches, H+ is released and detected. there is a semi conductor under each well for detection. they are natural nucleotides – don’t need to add anything. no lasers, cameras, fluorescence. quick and cheap. but not accurate. restricted bu the amount of data can collect. 10s of millions of reads. used in histology, IVF. specific systems for them.
single cell – 10k to 50k cells at same time. RNA expression. each cell in oil bubble. microfluidics. polymerase and nucleotides and gel beads. has dna with poly T tail. each strand has barcode. .collects mrna to it. lysis to cell. jelly barcode so know cell source of mrna. also sequence barcode for each strand UMI. no repetitions. mrna poly A attaches to poly t tail. all mrna of cell attaches.
applications – mrna, dna (methylation, histone modifications, etc) ,surface proteins (antibodies with barcode), intracellular proteins, can do mrna and surface proteins together on 10k cells.
solid tissue – single cell can change expression profile so needs to be quick. paraffin. 10 micron each slide. then analyse.
rna -> cdna -> amplification -> sequencing -> expression profiles
bulk NGS – all cells together. average expression.
single cell NGS – cell variation. isolate cells with mechanical or enzymatic. can enrich with FACS or magnetic bead based separation. need to isolate in oil droplets. microwells also capture individual cells. lyse cells. barcodes to see source cell. library preparation. amplified. loaded onto sequencer. data analysis and visualization.
use of single cell analytics – lineages, sup populations, novel genes, rare cell type.
10x genomes – makes oil bubbles. barcoded gel beads. cell and bead in bubble. collect. RT. pooling. remove oil. all cdna together. “hair” of gel bead, barcode of experiment, UMI, barcode, polyt. 10k cells. kits. software.
CITEseq – cell surface protein. barcode to each AB.
intracellular protein (PEA technology) – ABs with DNA tags bind to protein. dna hybridizes. specific. extend to amplicon. qPCR of 48 or 96 assays. 1536 assays by NGS. adaptors and barcodes. cluster for each protein. synthesis sequencing.
SMFISH – tissue ->fixation. probes coupled with flourophore. seen in microscope. in live. can see mrna moving.