lecture 8- NA sequencing

nucleic acid sequencing

process of determining the precise order of nucleotides on a DNA/RNA segment

targeted sequencing

identify one gene and read it

16s

marker gene in bacteria for targeted sequencing

ITS

marker gene in fungi for targeted sequencing

shotgun sequencing

study whole DNA/RNA

metagenome

study community structure and functional potential from community of DNA

metatranscriptome

studies community function of RNA

genomic

study genetic make up from single microbe

transcriptome

study gene expression of RNA from single microbe

sanger

only sequence one fragment at a time; high accuracy; slower and expensive; read long strands

next generation sequencing

millions of fragments can be sequenced; low accuracy; easy and cheap; read short strands

clone fragment in plasmid

what needs done for Sanger in amplification

add adaptors

what needs done for NGS in amplification

PCR

what is needed to start if you do amplicon sequencing

library preparation

first step in NGS; dna extraction, adding adaptors to fragments or PCR products

clonal amplification

second step in NGS; can be emulsion pcr or bridge pcr

cyclic array sequencing

third step in NGS

chemical cleavage reaction

not used today; used restriction enzymes but was only able to cut small fragments; gel would show the same sequence

dideoxy nucleotides

what does old Sanger method use to terminate fragments

pyrosequencing

library construction, clonal amplification via emulsion PCR, then sequencing as DNA is being synthesized

emulsion PCR

pcr happens in one bead

illumina

sequencing by synthesis

flow cell

where cluster amplification occurs in illumina

probes

what is solid sequencing based on

two

in sequencing by ligation, how many nucleotides are added per probe

semiconductor sequencing

pH change in wells

PacBio

uses SMRT technology, long read sequencing, circular consensus sequencing

nanopore

single, long molecule of DNA/RNA can be sequenced without PCR or chemical labeling; sequencing based on electrical current density