Sequencing by Synthesis Flashcards
Describe Illumina workflow (4)
- Sample prep - extraction and purification; requires a lot of DNA
- Library prep - adds special adapters recognized by sequencer
- Sequencing - reads sequence and creates raw data
- Analysis
How is DNA prepped for Illumina ?
- Mechanical fragmentation of genomic DNA; via acoustic sonication
-
End Repair & Phosphorylation
- starts with staggered ends
- enzymatic treatment = adds 5’ phosphate and blunts ends - Adenylation; to 3’ end = overhang
-
Adapter Ligation
- uses poly A overhang
- adapter attaches to either end of DNA
Aim of library prep
To obtain nucleic acid fragments with adapters attached on both ends
Which part of the adapter hybridizes to the lawn of the chip ?
P5 and P7
Which part of the adapter binds primers ?
Rd1 SP and Rd2 SP
How are libraries of Illumina quantified ?
- qPCR
- fluorometric methods
How are libraries of Illumina validated for quality ?
Bioanalyzer/ fragment analysis: should have a small, tight peak at the desired bp, in between lower and upper markers
Calculate library concentration
nM = ([Qubit or PicoGreen]* x10⁶) / (660 x library bp)
*NOTE: in ng/uL
What is a flow cell ?
- thick glass slide with channels or lanes
- each lane is coated with lawn of oligos complementary to library adapters
What is a cluster ?
thousands of copies of the same DNA strand positioned closely together
Describe DNA Cluster generation in Illumina
- ssDNA library hybridizes to primer lawn of oligos on flow cell
- DNA polymerase extends from 3’ oligo
- dsDNA molecule is denatured and original strand is washed away
REPEAT UNTIL LAWN OF OLIGOS ARE FULL:
4. Bridge amplification from P5 to P7
- DNA polymerase extends from 3’ oligo
5. Denature double-stranded bridge = doubles copies of ss templates
- In order to sequence, reverse strands are cleaved off = only forward strands left (P5)
NOTE: reverse strands (P7) are re-generated and sequenced after
Describe Sequencing by Synthesis (4) in Illumina
- Read 1 Primer hybridizes at the top
- Adds complementary fluorescent ddNTP = detected
- Chemically changes ddNTP to dNTP and cleaves fluorophore
- Continues synthesis one nucleotide at a time
Differentiate 4-channel vs 2-channel detection in
4-channel:
- uses one image
- each DNA base (GCAT) emits a different wavelength = 4 colours
2-channel:
- uses two images
G = no color (Gone)
C = red (Sea)
A = yellow (All)
T = green (Tea)
Explain Paired End Sequencing
Reads both ends of DNA:
- Forward strand = Read1 seq > cleave > i7 index seq
- Bridging and extension from P5 to P7
- Reverse strand = Read2 seq > cleave > i5 index seq
Differentiate the DNA insert and Index in Illumina adapters
DNA insert = different targets
- one long segment
Index = specific to patient sample
- Forward: i7 index seq AND Reverse: i5 index seq
Define Q Score
- “Phred quality score”
- assesses accuracy
- the unlikely probability that a given base is called incorrectly by the sequencer
T or F: a higher Q score is associated to better accuracy in Illumina
TRUE; a higher Q score is associated to better accuracy in Illumina
NOTE: Q score > 30 is ideal
What kind of output files is produced by Illumina ?
FASTQ
- Q scores encoded using ASCII characters (#?<:=)
Differentiate sequencing depth vs breadth
Depth: multiple reads in the same location of a sequence
- increased cost, but increased sensitivity & confidence
Breadth: more sequences in different locations are read
How is data analyzed in Illumina ?
- FastQ files produced
- reads are aligned to reference sequence = variants can be identified
In Illumina TruSeq Library preparation, what is the function of the polyadenylation step?
to facilitate ligation
What information is determined using a bioanalyzer trace in Illumina ?
the length of the sample DNA
