Sequencing and Bioinformatics Flashcards
Read length
of bases sequenced for a DNA fragment (provided as a maximum or mean)
Read depth
of times a nt is read during sequencing. A high read depth reduces errors
Reads per run
of sequences produced per run (usually provided as a maximum)
Accuracy (sequencing)
% error rate of an instrument (usually provided as 100% - error rate);
error includes substitition, base-specific bias, etc.
Time per run
Average time for a run in an HTS (High-Throughput Sequencing) instrument
Cost per million bases
Very variant across time and geographic region
Short-read instruments
300-500 bp reads (e.g. Illumina and Ion sequencing)
Long-read instruments
>50kbp (e.g. SMRT, Nanopore)
Pyrosequencing (Microfabricated picolitre reactors)
Emulsion PCR in droplets inside beads;
Known NT’s are flowed and washed away;
Optic slide sensor camptures emitted photons when a nt is incorporated;
1 M reads occur sumultaneously (i.e., parallel sequencing);
Sequences are put together as contigs (de novo);
96% - 98% accuracy
ion torrent (non-optical semiconductor-device)
Amplification occurs on beads inside wells;
Nucleotides are flowed step-wise;
Chip detects H ions released by DNApol upon incirporating nt’s as pH shift;
Reads occur simultaneously (i.e. parallel sequencing):
99.9% accuracy
Illumina sequencing
barcodes are placed on adaptors;
libraries prepared in 94 wells;
flow cell (glass slide) with lanes containing bound oligos;
bridge amplification (forward and reverse) repeated many times;
each nt has acgaracteristic fluorescence signal (sequencing through synthesis);
base call determined by wavelenght emission and signal intesnity;
Single-Molecule Real-Time (SMRT)
a single molecule is immobilized in a nanophotonic structure;
wavelenght is detected by feluorophore laser excitation;
each dNTP has a different fluorophore and emits a didferent color;
very fast and cheap
Single-molecule nanopore DNA sequencing (Nanopore)
Does not require sample amplification;
Does not require fluorescent labelling;
ssDNA molecule passes through a protein nanopore;
an adaptor on the membrane detects ionic current passing through pore;
each nt has a different ionic current;
Sagner sequencing (Manual Sanger dideoxy chain terminator DNA sequencing)
reactions happen inside microcapillaries;
fluorecent ddNTP (H instead of OH on 3rd C of ribose) makes amplification stop;
Laser excites fluorescin on each ddNTP;
Each ddNTP has a different wavelenght;
wavelength indicates at which nt amplification stopped;
output is electropherogram;
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