MIA FORA MFlex Operation and Software Training

Introduction to HLA

• HLA Concepts Review
• HLA Typing
• HLA Nomenclature

Introduction to NGS

• NGS Concepts Review
• Sanger Sequencing
• NGS Sequencing
• Whole Gene Approach
• NGS Comparisons and Advantages
• NGS Methodology
• NGS Instrumentation

Introduction to MIA FORA NGS MFlex HLA

• MIA FORA NGS MFlex HLA Typing Kit Solution
• MIA FORA NGS MFlex HLA Typing Kit Protocol
• MIA FORA NGS MFlex HLA Typing Kit Turnaround Time
• Introduction to MIA FORA Assay
• Manual Protocol
• Automated Protocol
• Create Project in MIA FORA NGS MFlex HLA Typing Software
• Long-Range PCR Overview
• Kit Components, Equipment
• Lab Sesseion – Lab Practicals Overview
• Practice 1: Analyze Quality of DNA Extracted from Blood
• Day 1 Wrap-Up

Library Construction

Introduction to Library Construction

• Library Construction Overview
• Fragmentation
• End Repair
• A-tailing
• Magnetic Bead Cleaning (Plate)
• Adaptor Ligation
• Pooling of Ligated Products
• Magnetic Bead Cleaning (Tube)
• Lab Practical Session
• Lab Practical Overview
• Practice 1: Primary Library Preparation
• Practice 2: Post Primary Library Preparation
• Practice 3: Index Adaptor Ligation
• Practice 4: Consolidation of Adaptor Ligated
• Products
• Practice 5: Magnetic Bead Cleaning (Tube)
• Day 2 Wrap-Up

Post Library Construction

• Day 2 Review
• Introduction to Post Library Construction
• Size Selection Overview
• Pippin™ Prep System
• Magnetic Bead-Based Size Selection
• Library Amplification
• Post Library Amplification Bead Clean
• Qubit™ Fluorometric Quantitation
• Quantitative PCR
• qPCR Calculations
• Kit Components
• Pippin Prep Reagent Kit
• Pippin Prep Gel Cassettes
• Qubit dsDNA Broad Range Assay Kit
• KAPA Library Quantification
• Equipment
• Pippin Prep System
• Qubit Fluorometer
• ViiA7™ qPCR System

Lab Practical Session

• Lab Practical, continued
• Practice 1: Size Selection using Pippin Prep
• Practice 2: Library Amplification
• Practice 3: Post Library Amplification Bead Cleaning
• Practice 4: Qubit Fluorometric Quantitation
• Practice 5: Qubit Calculations
• Day 3 Wrap-Up

Sequencing and Software Theory

Introduction to Sequencers

Preparing the Final Library for Sequencing

• Lab Practical Session:
• Sequencing Worksheets
• Practice 1: Preparing and Loading Sample on
• Illumina Sequencer
• Lab Practical, continued
• Introduction to Software Theory
• Background Information
• Sequencing/Sequencer
• Orthogonal Algorithms
• IMGT/Sirona Databases
• Coverage Metrics
• Day 4 Wrap-Up

MIA FORA Access and Reviewing Data

Data Review

• Accessing MIA FORA
• Desktop Environment Overview
• Log into MIA FORA
• MIA FORA Software Auxiliary Tools
• Connecting to the Software

Introduction to Activities and Reviewing Data

• Guided Software Activities
• Connect to the system
• Log In to MIA FORA
• Creating Users
• General Software Navigation
• Best Practices for Reviewing Data
• Class I Genes: A, B and C
• Class II Genes: DRB1,3,4,5, DPA/B, or DQA/B
• Novel Allele
• NULL Allele
• Data Review and Interpretation
• Reviewing User Data
• Report Formats
• Day 5 Wrap-Up