Samantha Court Genetic Technologist (Birmingham)

Transcription

1 National Genetic Technologist/Healthcare Science Practitioner Training Day Cardiff - 22 nd November 2017 Samantha Court Genetic Technologist (Birmingham)

2 ccf DNA is released from all cells undergoing apoptosis Quantity: 1-5 ng/ml of plasma Half-life: minutes Fragment size: ~166 bp

3 Dennis Lo et al during pregnancy a proportion of the ccfdna is derived from the fetus Fetal portion of ccfdna during pregnancy: 2-25% Detectable from 6-7 weeks gestation cffdna comprises shorter fragments of DNA ( bp) NON-INVASIVE PRENATAL DIAGNOSIS

4 Three year project funded by the Department of Health/Wellcome Trust Primary objective: Develop an assay for NIPD of Duchenne & Becker muscular dystrophies (DMD/BMD) & ensure it was cost effective for clinical application THE ASSAY: Non Invasive Prenatal Diagnosis (NIPD) by Relative Haplotype Dosage (RHDO) Targeted SNPs are sequenced by Massive Parallel Sequencing (MPS) using the Illumina MiSeq Instrument Tracks fetal inheritance of the mutated genes in a linkage-like manner

5 Secondary objective: Expand the testing repertoire to include other single gene disorders: Spinal muscular atrophy (SMA) Clinical feasibility: Universally applicable to most SGDs with potential to multiplex thus reduce costs Currently in validation: *Cystic Fibrosis (CF) * Congenital Adrenal Hyperplasia (CAH) Clinical service NIPD by Relative Haplotype Dosage (RHDO) Sept 2016 : Detection of DMD/BMD and SMA in cffdna

6 Maternal blood taken & received into lab Plasma Separation Cell free fetal DNA extraction using QIAsymphony Adapter Ligation & library amplification Pooling of sample libraries Hybridisation to capture probes Loading onto Illumina MiSeq for Sequencing ½ Day Full day Incubated Full day over weekend

7 Gestation of 8 weeks or above 20 ml Maternal blood taken into STRECK 5 days Plasma layer taken (8-10ml) & spun again (10 minutes) Sample sent to lab for processing Plasma Plasma aliquots stored at C Whole blood spun for 10 minutes to split into layers Buffy Coat Erythrocytes

8 Use the QIAsymphony DSP Virus/Pathogen Kit Load 4ml patient plasma Automated system takes 2.5 hours to run Process up to 12 samples per cartridge Produces reliable yields of 20 to 50ng of DNA

9 Samples Required Per Patient Maternal Plasma DNA (mat and fetal cfdna) Use all extracted Maternal Genomic DNA 100 ng Paternal Genomic DNA 100 ng Proband Genomic DNA 100 ng Can process three different patient batches per run

10 SAMPLE PREPARATION: DNA SHEARING: Shearing of genomic DNA samples is performed enzymatically using the KAPA HyperPlus prep kit END REPAIR AND A-TAILING: 5 3 AATGTACCATCAGCT GCCATTACATGGTA A-TAILING END REPAIR A 5 3 CGGTAATGTACCAT GCCATTACATGGTA 5 3 GCTATAAATCGCG CGATATTTAGCGC BLUNT ENDS

11 ADAPTER LIGATION: DNA ligase ligates adapters to A tail at either end of fragment Universal primer site Adenine Adenine Unique Index Sequence Universal primer site Bind to Flow Cell DNA FRAGMENT Bind to Flow Cell FORWARD ADAPTER REVERSE ADAPTER Sequencing Primer Site

12 POST ADAPTER LIGATION SAMPLE CLEAN-UP: Use Agencourt AMPure magnetic beads which bind to DNA Magnetic beads carrying DNA bind against magnetic strip on rack Supernatant is carefully removed from samples

13 Pre-capture Library Amplification (PCR): Amplification of DNA fragments with adapters correctly bound to both sides of the fragment. END OF FIRST DAY NGS PREP

14 Sample clean up Eluted in WATER Pre-Capture Quality Control checks: Ascertain how well the library prep process has worked. Qubit Fluorometer Yield for each sample should be > 500ng Tapestation Shows fragment sizes of the samples

15 GENOMIC DNA SAMPLES: Single peak at ~ 300bp Around 300bp peak corresponds to length of sheared DNA fragment (~200bp) plus ligated adapters

16 PLASMA cfdna SAMPLES: Two visible peaks ~300bp peak ~470bp peak ~115bp

17 Individual sample libraries are pooled together to a total mass of 1 µg of DNA. Pooled 2 parts cfdna to 1 part gdna

18 Use Biotinylated probes supplied by NimbleGen. Custom probe design ensures enrichment of specific regions of interest (SNPs targeted for analysis) Preparation: Want to ensure that probes hybridise at target sequences in patient DNA not to sequences on adapters SO. HE Index Oligos HE Universal Oligo COT DNA Pooled sample libraries

19 Preparation: Concentrate pooled sample in vacuum concentrator Evaporation Small volume of buffers added to rehydrate the DNA and then probe is added to the concentrated library Pooled Sample Hybridisation Probes

20 Probes will have bound to regions of interest within patient DNA over incubation period Streptavidin beads then added which bind to the probes bound to the DNA Non-specific DNA is washed away by using buffers of reducing stringency Stringency washes are temperature sensitive (maintain 47 o C)

21 Post-capture PCR: Enrichment of library consisting of target fragments which contain region of interest captured in previous step Target fragments enriched for sequencing Bead clean-up of enriched fragments for sequencing: Magnetic bead clean-up with two 80% washes as in previous steps Post-capture QC: Assessment of concentration & fragment size of DNA in final enriched/captured sample library

22 Final sample library is prepared for sequencing Correct dilution is critical to obtaining high quality & quantity of sequencing data Paired end sequencing run with 2 X 80 cycles Sequencing run overnight

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24 Assessment of overall quality of sequencing run QC Metric Illumina Guideline Ranges Cluster Density (K/mm2) Clusters Passing Filter > 80% Q30 Score > 85% Phas/PrePhas <0.2% Data passed to Bio-informatician /Scientist for analysis END OF TECHNICAL WORKFLOW

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26 DMD/BMD Total referrals 13 Affected 4 Unaffected 8 Suboptimal 1 SMA Total referrals 24 Affected 3 Unaffected 4 Unaffected carrier 14 Suboptimal 3

27 Michael Parks, Benjamin Bowns, Elizabeth Young, Samuel Clokie, Stephanie Allen & Julie Hewett Advanced Diagnostics, Genomics and Precision Medicine Award Winner West Midlands Celebration of Innovation Awards 2017