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Objectives
• Summarise the NGS assays currently available at BGL
• Look at the general challenges with NGS assays
• Highlight technical issues that could be encountered with specific
library preparation methods
– SureSelect
– HaloPlex
– TruSight
• Briefly consider future plans
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Current NGS assays
NGS Panel
Genes
Library prep
method
Clinical relevance
Inherited Peripheral
Neuropathies (IPN)
56
SureSelectXT
Genes associated with different types of inherited
neuropathy are tested to classify diverse phenotypes
Paediatric Cardiomyopathy (PC)
71
SureSelectXT
PC has multiple causes and can present in infancy with
cardiac failure and sudden death.
Chromosome Breakage
Disorders (CBD)
63
SureSelectXT
Diagnosis and classification of Fanconi anaemia and
related conditions characterized by a defect in DNA
repair mechanisms or genomic instability.
Inherited Bone Marrow Failure
Syndromes (IBMFS)
45
SureSelectXT
Classification of rare genetic blood disorders involving
aplastic anaemia associated with a family history.
Familial Hypercholesterolaemia
(FH)
5
HaloPlex
Caused by mutations in genes for cholesterol
metabolism. Premature atherosclerosis and increased
risk of CHD.
Osteopetrosis (OP)
21
HaloPlex
Diagnosis of very rare disorders characterized by an
increased bone density due to impaired bone resorption.
Aid decisions on potential curative stem cell transplants.
Steroid Resistant Nephrotic
Syndrome (SRNS)
37
HaloPlex
A disease of kidney filtration, resulting in massive and
unremitting protein loss into the urine. A confirmed
genetic diagnosis alters immunosuppressant treatment.
Cancer (BRCA/solid tumour
panel)
94
TruSight
Genes associated with common (e.g. breast, colorectal)
and rare cancers. 284 SNPs found to correlate with
cancer predisposition through GWAS.
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Current NGS assays
•
•
•
•
Currently all NGS assays are targeted gene panels looking for point mutations and
gene copy number variation, run on Illumina MiSeq
Agilent SureSelect XT Target Enrichment System
– 3µg input DNA (30ng/µl in 100µl)
– 5-6 days wet lab time
– Mechanical shearing by sonication using Bioruptor
Agilent Haloplex
– 200ng input DNA (5ng/µl in 45µl)
– 3 days wet lab
– Fragmentation by enzymatic restriction digestion
– Currently 16 indexed libraries pooled
Agilent TruSight
– 50ng input DNA (5ng/µl in 10µl)
– 4 days wet lab
– Enzymatic DNA fragmentation
– 24 indexed samples pooled
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NGS Considerations
•
•
•
•
Sample quantity/quality
– DNA extraction method
– Accurately quantify input DNA
• Two-step method of DNA normalisation is recommended
Labour intensive protocols; many liquid handling steps
– Confirm presence of product at appropriate intervals
– Sample transfer checks
– Reagent storage requirements
Data handling and management
– In-house pipeline with bioinformatician support
– Mass data storage requirement (Arkivum company pending)
Post-NGS work
– Follow-up primer design; Sanger sequencing to confirm mutations or fill gaps
• Provision for primer storage
• A system for location of primers for Sanger confirmations
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SureSelectXT
•
•
Generation of libraries from 3µg genomic DNA (relatively high input DNA)
– Request 5µg of DNA; at least 50ng/µl
• Allow for variation in DNA concentration between NanoDrop and Qubit
(fluorescence signal only when bound to target dsDNA)
• Allow sufficient DNA for confirmatory and follow-up procedures e.g.
Sanger sequencing and/or MPLA
• However, ~2µg DNA has worked well (IBMFS, IPN)
Fragmentation of DNA by sonication using a Bioruptor
– Optimum average starting fragment size 150-200bp
– Regularly achieved after 60 cycles (30 secs on/30 secs off)
– Inadequately sheared gDNA can result in suboptimal data
• Bioruptor LoBind tubes should be checked for abnormalities
• Diluted samples should not contain bubbles
– Chemagen-extracted DNA (beads) proved resistant to shearing.
• DNA clean-up + re-precipitation solved this problem
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SureSelectXT
•
•
•
24-hour 65°C hybridisation step
– Use a compression pad to minimise evaporation
Inefficient washing of post-hybridisation libraries can result in a high percentage
of off-target reads being maintained within the library preparation and loaded
onto the MiSeq
Residual AMPure beads from SureSelect protocol can give signal which runs with
the upper marker
– Any signal under the upper marker affects quantification
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Troubleshooting HaloPlex
• Mixed results; success, partial fail
and completely failed runs
• Highlights the importance of
complete QC data
•
•
•
•
•
Reagent log
Instrument calibration records
Regular equipment maintenance
Transfer checks
Use of controls
• Agilent supported
troubleshooting approach
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Troubleshooting HaloPlex
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Troubleshooting HaloPlex
• DNA samples
– Excess EDTA in DNA samples (present in TE buffer for eluting DNA)
can affect the activity of the restriction enzymes
• Reagents
– Replace NaOH made up in-house to buy in ready-made
– Dilute NaOH using larger volumes to ensure accuracy as the
concentration of 50mM NaOH to elute DNA is critical
– Ensure thorough mixing of pooled restriction-digested products to
ensure complete deactivation of restriction enzymes
• Carried ECD through whole process
• Reduce any possible bead carryover into PCR step
– Changed magnet
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TruSight
• New protocol in BGL
– TruSight Cancer assay available “off the shelf”, already optimised
– Initially BRCA1/BRCA2 only
– targets 94 genes suspected to play a role in predisposing to cancer
– associated with both common and rare cancers
• Samples pooled earlier in protocol
• Increased amounts of formamide
• Lower library prep concentrations than expected
– not affected data to date
Breast Cancer association
Breast and Ovarian Cancer
HNPCC – Hereditary non-polyposis colorectal cancer (Bowl Cancer)
Pheochromocytoma
- Tumor of the Adrenal gland
Neurofibromatosis - Tumours affecting the nerves
Tuberous sclerosis - Causes mainly benign tumours to grow in different parts of the body
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Future Developments
• Move from SureSelectXT to SureSelectQXT protocol
– Reduce time;
• No mechanical shearing
• Fragment DNA + adapter-tag DNA ends in single enzymatic step
– Less DNA;
• Only 50ng input gDNA (3µg with XT)
• Faster throughput
– Automation – a tender process for robot in progress
– Upscale number of libraries pooled and run on the MiSeq
• Exome sequencing
– Clinical exomes; larger panel testing using Nextera
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Thank you
Thank you to the BGL Sequencing Team
and
Thank you for listening! 
Exceptional healthcare, personally delivered
http://www.nbt.nhs.uk/genetics
Exceptional healthcare, personally delivered
http://www.nbt.nhs.uk/genetics
Exceptional healthcare, personally delivered
http://www.nbt.nhs.uk/genetics