How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization Kit - Swift Biosciences
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
How One NGS Core Lab Reduced Sequencing Costs with a
Novel Library Normalization Kit
GenomeWebinar
March 27, 2019
Julia Karow Tony Brooks Mida Pezeshkian
GenomeWeb UCL Genomics Swift Biosciences
Today’s Panelists
Julia Karow Tony Brooks Mida Pezeshkian
Managing Editor, Applications Specialist, Product Manager,
GenomeWeb UCL Genomics Swift Biosciences
(Moderator)
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization Kit
Please submit any questions in the Q&A panel
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization Kit
Widget Guide
There are a series of widgets to enhance your webinar experience in
the bottom tray of your window.
Help Slides Media Player Q&A Speaker Bio Resources
Can assist Displays the Plays the Use this to Learn more Download
if you are PowerPoint audio. You type in any about today’s available
having presentation. can use to questions presenters. handouts.
audio adjust you have
issues or the volume. for our
slides aren’t presenters.
advancing.
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization Kit
Tony Brooks
Applications Specialist,
UCL Genomics
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization Kit
Swift Normalase
How One NGS Core Lab
Reduced Sequencing
Costs with a Novel
Library Normalization Kit
Tony Brooks – Senior Sequencing Application Specialist
UCL Genomics Core Facility
→ Collaborative research core facility (research
and clinical)
→ Fully economically costed
→ Project design to analysis
→ Four dedicated applications specialists
→ >50,000 samples per annum
→ >700 individual projects per annum (100%
increase in 2yrs)
→ Latest genomic technologies and automation
→ Excellent partnerships and collaborations (ie
bioinformatics)
ichgenomics@ucl.ac.uk @uclgenomics http://www.ucl.ac.uk/ich/research/genetics-genomic-medicine/ucl-genomics
What does this mean in practice
35M reads per lane (1 Sample)
200-300M reads per lane (12-16 Samples)
10000M reads per lane (384 Samples)Quantification of libraries is important Absolute quantification Avoid over/under-load of flow-cell • Underload → Insufficient data / low sensitivity, poor consensus sequence & run failure • Overload → Poor base-call quality, low pass-filter, run failure
Quantification of libraries is important
Relative quantification
Avoid over/under sequencing samples in an experiment
8,000,000
n=30, CV = 26%
7,000,000
6,000,000
5,000,000
4,000,000
3,000,000
2,000,000
1,000,000
0
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30Methods for library quantification
Capillary Electrophoresis (Bioanalyzer / TapeStation)
+ Provides molarity (accounting for library size)
+ Detect presence of adaptor-dimer
- Slow (~30mins per 12 samples)
- Reproducibility?
- Inaccurate (±20% for TapeStation)Methods for library quantification
Dye-based quantification (Qubit)
+ Very accurate, even at low concentrations
+ Very reproducible
- Slow (~60mins per 96 samples)
- Doesn’t account for library size (gives mass/volume)
- No information about presence of dimerMethods for library quantification
qPCR
+ Very accurate, even at really low concentrations (“Gold-Standard?”)
+ Very reproducible
- Slow (~60mins per 24 samples (in triplicate) + 15 minutes analysis)
- Doesn’t account for library size
- Requires very accurate pipetting and mixing (standard curve)Pre-Normalase™ workflow – decision tree
Libraries
Qubit
Quantification
Normalization
TapeStation
Qubit Agreement
Quantification ±10%?
No Yes
Pool (based on
qPCR
Qubit or qPCR)
Qubit
Quantification
SequenceWetlab – Project Workflow by Time
Swift Normalase™
Full-length adaptors
R5 Reagent (modified P5/P7 primer mix) requires full-length adaptors
Illumina TruSeq LT/HT NEB Multiplex Oligos
IDT xGEN UMI/UDI adaptors Nextera (XT)
Kapa Single & Dual Indexes Agilent QXTPost-Normalase™ workflow – decision tree
Libraries
TapeStation QC
(Confirm >12nM)
Normalase I
Pool Equal
Volume (5µL
each)
Normalase II
Sequence
Example: 24 libraries in approximately 1 hourResults
6 Kapa mRNA Hyper Prep assays / IDT adaptors on HiSeq 3000 laneResults
(n=6) Kapa mRNA Hyper Prep / IDT xGen adaptors on HiSeq 3000 laneResults
(n=12) NEB Low-Input / IDT adaptors on Partial NextSeq run
AUTOMATED NORMALASE IResults
(n=32) Nonacus Cell3 Exome / NextSeq 500 RunResults
(n=4) Nonacus Cell3 Exome Pools / NextSeq 500 RunResults
Index hopping
Index 1 Index 2 Index 3 Index 4 Index 5 Index 6
Index 1 14.37 0.03 0.03 0.03 0.03 0.02
Index 2 0.05 17.53 0.06 0.04 0.03 0.03
Index 3 0.02 0.06 14.96 0.03 0.05 0.02
Index 4 0.03 0.04 0.08 16.32 0.03 0.11
Index 5 0.02 0.03 0.03 0.15 15.08 0.02
Index 6 0.03 0.02 0.02 0.03 0.04 13.49
Hopping rate: 1.20%
Undetermined: 7.06%
(n=6) Kapa mRNA Hyper Prep / HiSeq 3000 LaneSwift Normalase™
Saving Money
Without Normalase With Normalase
Qubit QC (n=24 @ $2/ sample = $48) TapeStation (n=24 @ $3/sample = $72)
TapeStation (n=24 @ $3/sample = $72) Time (1hr @ $100/hr = $100)
Time (4hrs @ $100/hr = $400) Normalase Reagents (n=24 @ $7.5/sample = $180
Total: $520 Total: $352
$21.67/sample $14.67/sample
$7 / sample savingSummary • Minimal hands-on time (≤ 10 minutes total) • Normalase I works with automation • Library balancing with typical CV < 10% • Final pool ready to load on sequencer • Removes the need to adjust for insert size • Compatible with multiple preps (Illumina / Kapa / NEB / Nonacus) • Low index hopping rates • Less chances manual of error due to exact same workflow for each library • Cost savings due to speed of processing
Mida Pezeshkian
Product Manager,
Swift Biosciences
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization KitQuestions?
Julia Karow Tony Brooks Mida Pezeshkian
GenomeWeb UCL Genomics Swift Biosciences
Please enter your questions in the Q&A panel on your screen.
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization KitThank you for your participation!
Please be sure to fill out our post-webinar survey
to let us know how we did!
GenomeWebinar March 27, 2019
How One NGS Core Lab Reduced Sequencing Costs with a Novel Library Normalization KitYou can also read
