all content ©2013 immucor, inc. program handouts/next generation... · •december 3rd, 2015 –...


Questions


• October 3rd, 2014 - Immucor, Inc. announced the collaboration with Sirona Genomics

- Stanford Genome Technology Center, Stanford University,

Palo-Alto, CA

• December 3rd, 2015 – MIA FORA HLA-NGS Product launched

• February 3rd, 2016 - CE Mark approval for MIA FORA

Immucor HLA-NGS Market Entry


MIA FORA delivers whole Gene NGS HLA typing

1. Complete sequence coverage of entire gene

• Eliminate assumption of uncovered sequence

• Identification of new alleles, including Null alleles

• Not applicable for exon based NGS

2. Provide results with no ambiguities in a single pass

• No secondary testing required to resolve questionable results

• Shorten turnaround time and reduce cost of testing by eliminating needs for additional testing

• Not applicable for exon based NGS

3. Provide high “Depth of Coverage” for accurate base calling

Depth of Coverage=The number of sequence read for a particular base

e.g. 100 X coverage means, on average, each base was sequenced 100 times


Why Illumina Platform?

• Most widely used NGS platform

• Very high sequence quality

(lowest error)

• Paired-end sequencing is built into the platform

• Multiple platforms give high flexibility – NextSeq and MiSeq

• Sample preparation is user-friendly

• Sample preparation protocols are independent of Illumina


SERIOUS

PRODUCTION SCALE! PRODUCTION SCALE

MANY SAMPLES | RUN

HIGH THROUGHPUT WGS | WES | T-OME

LOW THROUGHPUT TGRS | WGS (MICROBES)

PERSONAL SCALE FEW SAMPLES | RUN

NextSeq 500

NextSeq 500

HiSeq 2500

MiSeq

HiSeq X Ten

HiSeq 2500

Sequencing Power for Every Scale


Long Range PCR

Hands on time ~30min

Amplification ~6hrs

Quantification of PCR Products

Hands on time ~1hr

Gene Balancing

Hands on time ~1hr

Library Construction

Hands on time ~2hrs

Total time ~5hrs

Size Selection


Total time ~45min

Library Amplification


Total time ~1.5hrs

Library Quantification


Total time ~2hrs

Sequencing


Total time ~24hrs

Data processing

4hrs for 24 samples

Data Analysis

1hr for 24 samples

MIA FORA NGS Workflow

*Automation Protocols Available for Biomek 4000 (Beckman Coulter)*


Day 1 • Long range PCR (6hrs)

Day 2

• Gene Balancing (2hrs)

• Library Construction (5hrs)

Day 3

• Size Selection (45min)

• Library Amplification (1.5hrs)

• Library Quantification (2hrs)

Day 4-5

• MiSeq Run (24hrs)

• Data Processing (4hrs)

• Data Analysis (1hr)

From Sample to Results

MIA FORA NGS Turn Around Time


Sample Size

Up to 24 samples/kit to generate typing of 11 genes

Class I: HLA-A, -B, -C

Class II: HLA- DQA1, -DQB1, -DPA1, -DPB1, -DRB1,3,4,5

MIA FORA Kit Configuration


Kit Consists of Three Components

1. PCR module (ROU/CE)

• Nine tubes of “All-In-One” master mix to cover all 11 genes

• Single amplification condition for all 11 genes

2. Library module (RUO/CE)

• Reagents for Fragmentation, End Repair, A-Tailing, Adaptor Ligation, and Library Amplification

• Pre-aliquot Adaptor Index plate (24 indexes)

3. Software (RUO/CE)

• Standalone workstation with Pre-loaded software

MIA FORA Kit Configuration


MIA FORA PCR Module

Simple Assembly & One Cycling Condition for All Genes

Components

Competitor 1 Competitor 2 Immucor

Primer mix Primer Master Mix

Buffer Master mix DNA

dNTP mix Enzyme

*Q-solution DNA

**Enzyme

DNA

*Required for DQB1

**Different amount required for DQB

Cycling Conditions

Competitor 1 Competitor 2 Immucor

One condition for all genes

DQB1 requires separate cycling

condition

One condition for all genes


Low Drop-out Rate

• Unique gene balance program

– Minimizes amplification bias among different gene.

– Allows for earlier pooling of all loci creating a simpler assay with fewer technical errors


Product Details

15

• Low Throughput Type-LT • 24 SAMPLES

Run on a MiSeq platform 5 days

• High Throughput

• 4 plates 96 samples Run on a NextSeq platform 7 days


Automation with Beckman Coulter Biomek FX Pre- and post-PCR


Software Features

o Extended Sequence Analysis

• Software provide analytical redundancy (3 algorithms) to accurately confirm allele assignment

• Exceeding ASHI requirement

o Haplotype Identification

• MIA FORA NGS achieves a high level of resolution at allele level

• Provide accurate haplotype identification

• Flag novel haplotype for further analysis


Product Availability

• MiSeq

– December 2015 RUO available for US

– January 2016 CE mark in Europe

– Canadian License after CE marking in 2016

• NextSeq – Summer 2016


Conclusions

Why MIA FORA?

– All-in-One PCR master mix for simple assembly

– Single Cycling Condition for all genes

– Proprietary PCR mix to prevent allele drop out

– Gene Balancing program for balanced allele coverage

– Highest coverage for both class I and class II genes

– Short hands on time

– Almost zero ambiguities

– Three algorithms for the most accurate base calling

and allele assignment


Continuing Education

• Each complete Attendance Roster:

– https://www.surveymonkey.com/r/6STH7FV

• Registration deadline is February 26, 2016

• Certificates will be sent via email only to those who have registered by March 15, 2016

• No registration for CE will be accepted after February 26, 2016

https://www.surveymonkey.com/r/6STH7FV


• PACE/California DHS

– 437-301-16

• Florida BPR

– 20-540134

• ABHI CECs: 0.225

– 1.5 Contact Hours

• Attendance Roster:

https://www.surveymonkey.com/r/NGSweb

Continuing Education

https://www.surveymonkey.com/r/NGSweb


Questions?

Please also feel free to email questions to Carly Callender: [email protected]


Thank you!

all content ©2013 immucor, inc. program handouts/next generation... · •december 3rd, 2015 –...

Documents