target enrichment sequencing - pacbio
TRANSCRIPT
For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved.
June.27.2017 / http://programs.pacificbiosciences.com/l/1652/2017-03-25/3sn5p2
PacBio Americas User Group Meeting Sample
Prep Workshop Breakout Session:
Target Enrichment Sequencing
AGENDA
Introduction
-Overview of Target Enrichment Applications and Methods
-Barcoding Options for Target Enrichment
-PCR-Based Target Enrichment
-Probe-Based Target Capture Enrichment (With PCR)
-Target Capture Enrichment Without Amplification
-Technical Resources for Target Enrichment Sequencing
PacBio Scientific Conference Poster Presentations
Q&A and Open Discussion
Overview of Target Enrichment
Applications and Methods
EXAMPLES OF TARGET ENRICHMENT USE CASES
(HUMAN BIOMEDICAL RESEARCH)
Image source: http://worms.zoology.wisc.edu/zooweb/Phelps/ZWK99004k.jpeg 4/6/15
5 Mb contiguous region
of chromosome 6
5 entire genes on 5
different chromosomes
All exons in one gene
on chromosome 5
- Also applies to targeting respective full-length cDNAs
VARIANT DISCOVERY AND DETECTION FOR ANY GENOME,
ANY REGION
Cost-effective target enrichment workflows providing accurate, unbiased
results
- Complete and uniform coverage for your targets, even in low complexity regions
- Repeat expansions
- Promoters
- Flanking regions of transposable elements
- Characterize the full spectrum of genetic variation
- SNPs
- Structural variation
- Indels
- Alternative transcripts
- Low-frequency variants
- Haplotypes
Human Biomedical
Research
Plant and Animal
Sciences
Microbiology and
Infectious Disease
COMMON TARGET ENRICHMENT METHODS
PCR-BASED
(no capture)
ISOTHERMAL
AMPLIFICATION
PROBE-BASED
CAPTURE (w/PCR)
NO
AMPLIFICATION
Select SMRTbell
Templates of Interest
Make SMRTbell
templates from gDNA
Solutions Available Evaluating Partners Late-Stage
Development
TARGET ENRICHMENT METHODS HAVE STRENGTHS & WEAKNESSES
PCR-BASED
AMPLIFICATION
(NO CAPTURE)
PROBE-BASED CAPTURE
(w/PCR)NO AMPLIFICATION
STRENGTHS
- Less complex
- Fast (<1 day)
- Low input DNA
- Low upfront cost
- Quickly iterate optimizations
- Probe-design flexibility
- Large target size (Mb)
- Large fragments (≥6 kb)
- Ability to phase larger regions
- Maintains DNA mods
- Maintains repeat length
- No PCR artifacts
WEAKNESSES
- Difficult to amplify repeats
- Lose ability to detect DNA mods
- Limited total target size
- Designing multiple primers
- More complex workflow
- Longer workflow
- Higher upfront costs
- Lose ability to detect DNA mods
- More complex workflow
- Higher input DNA
KEY APPS /
TARGETS
- Fewer, longer amplicons
- Many, shorter amplicons
- Larger contiguous regions (10s
of kb to multiple Mb)
- Maintains DNA mods &
repetitive regions
TECHNOLOGIES /
KITSGenDx (HLA), Amplicon Panels
(Multiplicom)NimbleGen, IDT, SureSelect CRISPR-Cas9
APPLICATION
EXAMPLESHLA, 16S, CYP2D6, Variant
Confirmation
Cancer Panel, MHC Region,
Several Genes
Nucleotide Repeat
Expansions, Methylation
Barcoding Options for Target
Enrichment
BARCODES ALLOW TARGETED SEQUENCING TO BE MORE
COST-EFFECTIVE
TARGETED
SEQUENCING
WITHOUT BARCODES
Multiple Samples
One Library Prep
One SMRT Cell
$
TARGETED
SEQUENCING WITH
BARCODES
Multiple Samples
Multiple Library Preps
Multiple SMRT Cells
$$$
OPTIONS TO INCORPORATE BARCODES IN WORKFLOW
Adding the barcodes earlier in the template prep process allows
earlier sample pooling, saving time and reagents
Add Barcodes
BEFORE or DURINGAmplification
Add Barcodes
AFTER Fragmentation
or Amplification
Genomic DNA
Amplification or Fragmentation
PACBIO BARCODING OPTIONS FOR TARGETED SEQUENCING
BARCODED UNIVERSAL PRIMERS
BARCODED HAIRPIN ADAPTERSLOCUS-SPECIFIC PRIMERS
TAILED WITH BARCODES
LINEAR BARCODED ADAPTERS
PCR
PCR
Ligation
Fragment
Ligation
Order from Third Party (12 barcodes)
Order from Third Party (384 barcodes)
Product (96 barcodes)
Product (96 barcodes)
INCORPORATION THROUGH PCR AMPLIFICATION INCORPORATION BY LIGATION
PCR
PAIRING TARGET ENRICHMENT & BARCODING METHODS
PCR-BASED
AMPLIFICATION
(NO CAPTURE)
PROBE-BASED
CAPTURE
(w/ PCR)
CAS9 CAPTURE
NO AMPLIFICATION
Barcoded
Universal Primers
RECOMMENDEDrequires 2-step PCR;
less expensive upfront primer
development
Not Applicable Not Applicable
Locus-Specific
Primers Tailed
with Barcodes
RECOMMENDEDgood for high-volume assays;
more expensive upfront to make
primers
Not Applicable Not Applicable
Linear Barcoded
AdaptersNot Applicable RECOMMENDED Not Applicable
Barcoded
AdaptersNot cost-effective Not cost-effective
LIKELY TO BE
RECOMMENDED(still in development)
PCR-Based Target Enrichment
COMMON TARGET ENRICHMENT METHODS
PCR-BASED
(no capture)
ISOTHERMAL
AMPLIFICATION
NO
AMPLIFICATION
Select SMRTbell
Templates of Interest
Make SMRTbell
Templates from gDNA
Solutions Available Evaluating Partners Late-Stage
Development
PROBE-BASED
CAPTURE (w/PCR)
PACBIO BARCODES
-Set of 384 barcodes (16 bp length), optimized for SMRT Sequencing
POOL BARCODED
AMPLICONS
1 SMRTBELL
LIBRARY PREP
SEQUENCING ON
1 SMRT CELL
ANALYSIS FOR
EACH BARCODE
PRIMER DESIGN
Target of Interest , Sample #1
Barcode #1
Barcode #1
Target of Interest, Sample #2
Barcode #2
Barcode #2
X hundreds of
targets & samples
Mandelker et al. (2016) Genet Med 18: 1282-1289
SHORT READ SEQUENCING PLATFORMS HAVE A DIFFICULT
TIME DIFFERENTIATING GENES FROM THEIR PSEUDOGENES
PMS2 EXAMPLE
- The PMS2 gene is associated with autosomal dominant Lynch syndrome (also called hereditary nonpolyposis colorectal cancer syndrome, or HNPCC)
- Identifying variants in PMS2 is hampered by the presence of a pseudogene, PMS2CL, which has nearly identical homology to PMS2 in the final four exons of the gene (exons 12–15)
- 99.2% identical (exons) vs. 98.2% identical (gene)
- Sequence reads derived from hybridization capture and short read sequencing methods cannot be unambiguously aligned to PMS2 or PMS2CL
PMS2
PMS2CL
10 11 12 13 14 15
1 2 3 4 5 6
98.2% identical
TARGETING APPROACH – LONG RANGE PCR
-This generates a ~17 kb amplicon that can be turned into a SMRTbell
template and sequenced on a PacBio sequencing system
PMS2
PMS2CL
10 11 12 13 14 15
1 2 3 4 5 6
Design primers so that only PMS2 will amplify
17 kb amplicon from PMS2
-After making a library and sequencing, data is run through Long Amplicon
Analysis
-Detection of all variants (exonic & intronic)
-Results in fully phased haplotypes:
Mandelker et al. (2016) Genet Med 18: 1282-1289
wt
mut
5 kb
PMS2 RESULTS
SIMILAR GENE EXAMPLES
Gene Disease
PMS2 Lynch syndrome/hereditary nonpolyposis colorectal cancer
PKD1 Polycystic kidney disease
SMN1 Spinal muscular atrophy
SDH Hereditary paraganglioma-pheochromocytoma syndrome
VHL von Hippel-Lindau (VHL) disease
MECP2 Rett syndrome or other MECP2-related disorders
FLCN Birt-Hogg-Dube syndrome
CFTR Cystic fibrosis
BRCA Hereditary breast and ovarian cancer
GRHP Primary hyperoxaluria type 2
HHT, ENG, ACVRL1 Hereditary hemorrhagic telangiectasia, types 1 and 2
NPC Niemann-Pick type C
MLYCD Malonyl-CoA decarboxylase deficiency
AGXT Primary hyperoxaluria type 1
CYP21A2 21-hydroxylase deficient congenital adrenal hyperplasia
Probe-Based Target Capture
Enrichment (With PCR)
COMMON TARGET ENRICHMENT METHODS
PCR-BASED
(no capture)
ISOTHERMAL
AMPLIFICATION
NO
AMPLIFICATION
Select SMRTbell
Templates of Interest
Make SMRTbell
Templates from gDNA
Solutions Available Evaluating Partners Late-Stage
Development
PROBE-BASED
CAPTURE (w/PCR)
PROBE-BASED TARGET CAPTURE ENRICHMENT PROTOCOLS FOR
MULTIPLEXED SAMPLES
http://www.pacb.com/wp-content/uploads/Procedure-Checklist-Target-
Sequence-Capture-Roche-NimbleGen-SeqCapEZ-Library-
PacBioBarcodedAdapters.pdf http://www.pacb.com/wp-content/uploads/Unsupported-Protocol-
Target-Sequence-Capture-Using-IDT-Library-PacBio-Barcoded-
Adapters.pdf
PACBIO BARCODED LINEAR ADAPTERS ENABLE MULTIPLEXING WITH
NIMBLEGEN SEQCAP® EZ AND IDT TARGET CAPTURE LIBRARIES
Illumina Barcodes
PacBio Barcodes
gDNA
gDNA
Univ.Seq Barcode Univ.SeqBarcodegDNA
gDNAUniv.Seq Barcode Univ.SeqBarcode
Univ.Seq Barcode Univ.SeqBarcodegDNA
gDNAUniv.Seq Barcode Univ.SeqBarcode
Univ.Seq Barcode gDNA
gDNAUniv.Seq Barcode Illumina barcode
Illumina barcode
Univ.SeqBarcodegDNA
gDNA Univ.SeqBarcodeIllumina barcode
Illumina barcode
Adapter Ligation PCR (Pre-SMRTbell prep)
Univ.Seq Barcode Univ.SeqBarcodegDNA
gDNAUniv.Seq Barcode Univ.SeqBarcode
• 6 bp Illumina barcode
• Asymmetric design – Barcode only on one
end (not accurately identified)
• Not amenable to PacBio data workflow
• 16 bp PacBio barcodes
• Symmetric design - Barcode on both ends
enables accurate identification
• Compatible with PacBio data workflow
PACBIO MULTIPLEXED TARGETED PROBE-BASED CAPTURE
WORKFLOW
Shear to 7 kb AmplificationProbe hybridization,
Bead capture, Wash
EXPERIMENTAL PIPELINE
INFORMATICS PIPELINE
Phasing with
SAMtools
Bin reads by
haplotype
Quiver
haplotypes
Tertiary
Analysis
Map Reads of
Insert to Hg19
1 2 3 4 5
9 10 11 12 13
Size Selection
3
5-9 kb
5-9 kb
6
Amplification and
SMRTbell prep.
+ Size Selection78
SequencingAnalysis
Genomic DNA
Ligate
Barcoded
Adapters
https://github.com/PacificBiosciences/targeted-phasing-consensus
OVERVIEW: PACBIO PROBE-BASED CAPTURE OPTIONS
DNA probes DNA probes RNA probes
Complete Probe Tile Flexible Probe Design Under Evaluation
Flat rate up to 7 MB Cost per probe
Predesigned & Custom
Panels
Predesigned & Custom
Panels
Faster Turnaround
Sequence data generated on PacBio RS II and MiSeq from cell line NA12762 captured with standard NimbleGen Oncology Panel
TRUE FULL-GENE ANALYSIS
-Example: MUTYH
PacBio
(~5 kb
fragments)
MiSeq
(200 bp
fragments)
Sequence data generated on PacBio RS II and MiSeq from cell line NA12762 captured with standard NimbleGen Oncology Panel
PHASING VARIANTS OVER LARGE DISTANCES
-BRCA1, exon 10:
PacBio
(~5 kb
fragments)
MiSeq
(200 bp
fragments)
Allele 1
reads
Allele 2
reads
Sequence data generated on PacBio RS II and MiSeq from cell line NA12762 captured with standard NimbleGen Oncology Panel
RESOLUTION OF STRUCTURAL VARIATION
-PDE4DIP:
PacBio
(~5 kb
fragments)
MiSeq
(200 bp
fragments)
Allele 1
reads
Allele 2
reads
PacBio resolves a heterozygous ~740bp deletion containing an entire exon,
missed by Illumina
-Combining gDNA and cDNA sequencing data gives better insight into how
DNA variants effects gene expression
-Captured and sequenced 35 AD candidate genes in two AD patients
• Average gDNA fragment size: ~6 kb
• Full-length transcripts ranging from <1 kb - ~10 kb
APPLYING LONG-READ SMRT SEQUENCING FOR VARIATION
SCREENING IN ALZHEIMER’S DISEASE
SNPs AND LARGER SVs DISCOVERED IN THE AD SAMPLES
Results:
-Detected a broad range of
genomic variants:
- In addition to SNPs, 31 unique
SVs were observed ranging
from 65bp to several kb in size
-515 and 507 total isoforms
found for patients 1 and 2,
respectively
-Only 39 were shared among
samples and Gencode v25
database
67 2
339
319
154
312
RIN3 GENE: ~50 bp INSERTION DETECTED
APP GENE: ~550 bp INVERSION DETECTED
~550 bp inversion
Allele 1 (5 isoforms)
Allele 2 (21 isoforms)
MAPT GENE: 26 TRANSCRIPT ISOFORMS DETECTED
-Novel exon (red arrows) found is 3 of the 5 isoforms in Allele 1. Not
observed in any isoforms from Allele 2.
MAPT gene results:
- Detected a
heterozygous
deletion
- One allele is
transcribed into
21 isoforms while
the other to only 5
- Detected a novel
exon and
transcripts
MAPT GENE RESULTS FOR SUBJECT 1
21 isoforms
5 isoforms
Heterozygous SNPs can be used to phase gDNA and transcripts into multi-kilobase
long haplotypes
Target Capture Enrichment
Without Amplification
REPEAT EXPANSION DISEASES
37
CRISPR/CAS9 SYSTEM
Some in vivo applications:
- Gene silencing
- Homology-directed repair
- Transient gene silencing or transcriptional repression
- Transient activation of endogenous genes
- Transgenic animals and embryonic stem cells
• Bacterial Adaptive Immunity
• RNA-guided DNA Endonuclease
PCR-FREE TARGET ENRICHMENT VIA CAS9 DIGESTION:
METHOD OVERVIEW (CURRENTLY IN DEVELOPMENT)
DETAILED CAS9 METHOD
1. Complexity Reduction – Digest gDNA with restriction enzymes to remove 80% of genome
2. Cut with EcoRI and BamHI; make standard SMRTbell library; Exo Digestion
3. Cut open specific SMRTbells with Cas9; Ligate PolyA Hairpin
4. Capture PolyA-containing SMRTbells on Magnetic Bead; Elute off Bead
5. Anneal primer; Complex with Polymerase;
Magbead Load onto SMRT Cell; Sequence
USING CRISPR/CAS9 TO ENRICH FOR REPEAT EXPANSION
DISORDERS
- Improved on-target rate with complexity reduction:
- Restriction enzymes are used to degrade unwanted SMRTbell templates
- Additional starting DNA is required to maintain input into Cas9 digestion step
-Multiplexing:
-Multiple regions can be targeted in the same reaction
- Patient samples could be barcoded during initial SMRTbell library preparation
Number of individual molecules sequenced
*
*
*Restriction Enzyme
COVERAGE ACROSS THE GENOME
CAS9 – “ON TARGET” READS
Target
HUNTINGTON’S DISEASE (HD)
-Autosomal dominant neurodegenerative
genetic disorder
-Caused by an expansion of a CAG triplet
repeat stretch in the Huntingtin (HTT) gene
- polyglutamine tract
CAG REPEAT COUNTS
CAG REPEAT COUNTS IN HT PATIENTS
Samples obtained from Vanessa
Wheeler (Harvard Medical School)
-Widening repeat number distribution
at the mutated allele is biological
- Obtained roughly equal number of
sequenced molecules for normal and
mutated alleles
FRAGILE X SYNDROME
-Most common heritable form of cognitive impairment
-Caused by expansion of a CGG trinucleotide repeat in the 5’ UTR of the
FMR1 gene
fraxa.org
>700 CGG REPEATS SEQUENCED FROM THE FMR1 GENE
• Difference in risk is greatest
near 75-80 CGG repeats
• Having full sequence
information is medically
relevantgollen et a
AGG “INTERRUPTIONS” REDUCE THE CHANCES OF PRE- TO FULL-
MUTATION TRANSMISSION
2 …CGG CGG CGG CGG AGG CGG CGG CGG CGG CGG CGG CGG CGG CGG AGG CGG …1 …CGG CGG CGG CGG AGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG …0 …CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG …
Yrigollen et al. (2012) Genet Med 14:729–736
…CGG CGG CGG CGG AGG CGG…
80%
60%
15%
DIRECT DETECTION OF METHYLATION
METHYLATION DETECTION OF FMR1 SAMPLE
METHYLATION DETECTION OF FMR1 SAMPLE
• CGG repeat region appears to be heavily methylated (5mC)
CONCLUSION
-Target any hard-to-amplify genomic region regardless of sequence context
-Avoid PCR bias and PCR errors
-Accurately sequence through long repetitive and low-complexity regions
- Count repeats and identify sequence interruptions
-Detect and characterize epigenetic modification signals
- Detect sample mosaicism
Amplification-free enrichment with CRISPR/Cas9 and SMRT Sequencing
achieves the base-level resolution required to understand the underlying
biology of repeat expansion disorders
Technical Resources for Target
Enrichment Sequencing
User Bulletins
User Bulletin for PacBio RS II and Sequel Systems: Centrifuge Tube and Pipet Tip
Recommendations (NEW!) (May 2017)
- PacBio advises against the use of Axygen MAXYMum Recovery™ tubes and pipet tips.
Please discontinue use of these products immediately. PacBio recommends alternatives
in the User Bulletin.
- http://www.pacb.com/wp-content/uploads/User-Bulletin-Centrifuge-Tube-and-Pipet-Tip-
Recommendations.pdf
Field Advisory for Sequel System: Securing Sequel Pipet Tip Rack (NEW!) (May 2017)
- PacBio recommends a simple procedure to ensure that the Sequel Pipet Tip rack is
firmly affixed to the tip box.
- http://www.pacb.com/wp-content/uploads/Field-Advisory-Notice-Securing-Sequel-Pipet-Tip-
Rack.pdf
User Bulletin for Sequel System: Heat Seal Advisory (Adhesive Seal Warning) (NEW!) (May 2017)
- PacBio advises against the use of adhesive foils and recommends the use of Sequel Sample
Plate Foil.
- http://www.pacb.com/wp-content/uploads/User-Bulletin-Heat-Seal-Advisory-Adhesive-Seal-
Warning.pdf
User Bulletin for Sequel System: Barcode Scanning of Sequel Sequencing Kit 2.0 (NEW!) (May
2017)
- PacBio is providing clarity on which barcode to scan to ensure the Sequel System has the correct
information and that all the consumables are compatible.
- http://www.pacb.com/wp-content/uploads/User-Bulletin-Barcode-Scanning-of-Sequel-Sequencing-
Kit-2.0.pdf
Find all protocols at http://www.pacb.com/support/documentation/
TECHNICAL RESOURCES FOR TARGET CAPTURE ENRICHMENT
Genomic DNA Target Sequence Capture Protocols
Shared Protocol – Target Sequence Capture Using Roche NimbleGen SeqCap EZ Library (2015)
- http://www.pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Target-Sequence-Capture-
Using-NimbleGen-SeqCap-EZ-Library.pdf
Procedure & Checklist – Target Sequence Capture Using SeqCap EZ Libraries with PacBio
Barcoded Adapters (NEW!) (May 2016)
- http://www.pacb.com/wp-content/uploads/Procedure-Checklist-Target-Sequence-Capture-Roche-
NimbleGen-SeqCapEZ-Library-PacBioBarcodedAdapters.pdf
Unsupported Protocol – Target Sequence Capture Using IDT Library with PacBio Barcoded
Adapters (NEW!) (January 2017)
- http://www.pacb.com/wp-content/uploads/Unsupported-Protocol-Target-Sequence-Capture-
Using-IDT-Library-PacBio-Barcoded-Adapters.pdf
Full-length cDNA Target Sequence Capture Protocols
Shared Protocol – Full-length cDNA Target Sequence Capture Using SeqCap® EZ Libraries
- http://www.pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Full-length-cDNA-Target-
Sequence-Capture-Using-Roche-NimbleGen-SeqCap-EZ-Library.pdf
Unsupported Protocol – Full-length cDNA Target Sequence Capture Using SeqCap® EZ Libraries
- http://www.pacb.com/wp-content/uploads/Unsupported-Protocol-Full-length-cDNA-Target-
Sequence-Capture-IDT-xGen-Lockdown-Probes.pdf
Application Notes
Multiplex Target Enrichment Using Barcoded Multi-Kilobase Fragments and Probe-Based Capture
Technologies (NEW!) (2016)
- http://www.pacb.com/wp-content/uploads/multiplex-target-enrichment-barcoded-multi-kilobase-
fragments-probe-based-capture-technologies.pdf
Targeted sequencing on the PacBio RS II using the Roche NimbleGen SeqCap EZ system
- http://www.pacb.com/wp-content/uploads/2015/09/Application-Note-Targeted-Sequencing-on-the-
PacBio-RS-II-Using-the-Roche-NimbleGen-SeqCap-EZ-System.pdf
Data Analysis
Data Analysis Workflow for Haplotype Phasing of Heterozygous SNPs (Support for phasing and
generating consensus sequence with SAMtools)
- https://github.com/PacificBiosciences/targeted-phasing-consensus
WHERE TO FIND SMRT RESOURCES
http://www.pacb.com/smrt-science/smrt-resources/
Explore our collection of resources and learn how scientists use SMRT Sequencing to advance their research.
Scientific publications
Explore our database of scientific publications featuring PacBio data.
Conference proceedings
Access conference posters and presentations our customers, collaborators, and internal scientists have presented at
various scientific meetings.
PacBio literature
View case studies, brochures, application notes, and more.
Video gallery
Watch our collection of videos, webinars, customer testimonials, and more.
Blog
Read our blog featuring new research, publications, conference summaries, and SMRT Sequencing updates.
Product documentation and training
Visit user documentation for our entire documentation library and training for user training materials.
PacBio Scientific Conference
Poster Presentations
http://www.pacb.com/wp-content/uploads/Kujawa-AGBT-2017-Alzheimers-Disease-Candidate-Genes-
and-Transcripts-Using-Hybridization-Capture.pdf
AGBT 2017
http://www.pacb.com/wp-content/uploads/Ekholm-AGBT-2017-Screening-and-characterization-of-
causative-structural-variants-for-bipolar-disorder.pdf
AGBT 2017
Poster PDF Available Upon Request
ESHG 2017
http://www.pacb.com/wp-content/uploads/Clark-AGBT-2017-Targeted-SMRT-Sequencing-of-Difficult-
Regions-of-the-Genome-Using-a-Cas9-Non-Amplification-Based-Method.pdf
AGBT 2017
Q&A and Open Discussion
Q&A AND OPEN DISCUSSION
Target Capture Enrichment with Roche NimbleGen’s SeqCap EZ Technology
Q: How does the target enrichment protocol work?
A: Customers order an off the shelf exome, human and non-human designs or custom designs and reagents from Roche NimbleGen using the Target
SeqCap EZ shared protocol, and perform standard SMRTbell® template preparation.
Q: What target region can be used?
A: Human and non-human genomic regions and exomes are supported by the SeqCap EZ technology.
Q: What are the different types of enrichments that are available?
A: There are 4 types of SeqCap EZ libraries:
- SeqCap EZ Exome – variants of human exomes
- SeqCap EZ Choice – customer specified subsets of human probes
- SeqCap EZ Design – off-the-shelf design of human (e.g., MHC) and non-Human (e.g., soybean exome)
- SeqCap EZ Developer – completely custom design
Q: What organisms are compatible with this target enrichment approach?
A: All organisms supported by the SeqCap EZ technology.
Q: Does Roche NimbleGen support this protocol?
A: Yes, any design and target procedure questions should be directed to Roche NimbleGen. Any SMRTbell library preparation questions should be
directed to PacBio.
Q: What results have been achieved with this target enrichment approach?
A: To date, we have evaluated the ‘off-the-shelf’ Major Histocompatibility Complex (MHC) and Comprehensive Cancer panel kits. Example results can
be viewed in the Application Note: Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture
technologies (http://www.pacb.com/wp-content/uploads/multiplex-target-enrichment-barcoded-multi-kilobase-fragments-probe-based-capture-
technologies.pdf) and PacBio’s AGBT 2015 Poster: Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ
enrichment (http://www.pacb.com/wp-content/uploads/Poster_TargetedSMRTSequencingPhasing_RocheNimbleGenSeqCapEZ.pdf)
Frequently Asked Questions – Target Enrichment
Q&A AND OPEN DISCUSSION
Q: How long are the enriched fragments?
A: Genomic DNA is fragmented to 10kb. After size selection, capture, amplification and SMRTbell® library preparation, the average insert size is
approximately 6 kb.
Q: What is typical length of fragments targeted with the SeqCap EZ technology?
A: In the SeqCap EZ protocols, the DNA is sheared to the appropriate length for the sequencing technology.
Q: What is the advantage of the longer 6kb fragments?
A: The PacBio® System often delivers even coverage over multi-kilobase regions of the genome. With PacBio long reads, heterozygous SNPs can be
used to phase the reads and generate accurate haplotypes. It also provides good coverage for intronic and exonic regions across the target of
interest.
Q: For the MHC work described in PacBio’s AGBT 2015 Poster: Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap
EZ enrichment (http://www.pacb.com/wp-content/uploads/Poster_TargetedSMRTSequencingPhasing_RocheNimbleGenSeqCapEZ.pdf)
- How did PacBio confirm the SNPs and haplotypes?
- What was the level of enrichment achieved compared to gDNA?
- What was the percent of reads that were on target?
- How did you phase/haplotype the reads?
A: For the MHC work described in the AGBT 2015 Poster
- Data from the MHC target enrichment experiment was compared to data generated from sequencing with both the PacBio System using
amplicons with the GenDx NGSgo®-AmpX amplification primers and traditional Sanger methods.
- On average the enrichment was >1500-fold for the comprehensive panel and >600-fold for the MHC panel.
- On average the percentage of reads on target was >60%.
- For each targeted region, SAMtools was used to phase and bin reads by haplotype, and then Quiver was applied to polish each haplotype to
high consensus accuracy. This entire workflow is summarized on GitHub here
Q: Where can I find more information about Target Capture Enrichment?
A: Please visit the Targeted Sequencing section of PacBio’s website (http://www.pacb.com/applications/targeted-sequencing/)
Frequently Asked Questions
Frequently Asked Questions - General
How long can I store my polymerase-bound sample?
- PacBio RS II:
- PacBio recommends that polymerase-bound samples be stored at 4C and used within 3 days.
- Sequel System:
- PacBio recommends that polymerase-bound samples be stored at 4C and used within 7 days.
How do I dissociate my polymerase-bound sample from MagBeads?
- Dissociating polymerase-bound sample from MagBeads may damage the sample and is not recommended. PacBio recommends binding
sample to MagBeads immediately before sequencing and proceeding with sequencing as soon as possible. If a delay between MagBead
binding and sequencing is unavoidable, Customers can store the sample in the dark at 4C, but delaying sequencing will be at the
Customer’s own risk. If a MagBead sample has already been aliquoted into a sample plate, the sample plate should be sealed upon storage
at 4C. For Sequel samples, the sample plate should be heat-sealed with the Sequel Sample Plate Foil (P/N 100-667-400). For PacBio RS II
samples, the sample plate should be temporarily sealed with an adhesive microplate sealing film and then the sealing film should be
replaced with the PacBio RS II Sample Plate Septum (P/N 000-882-901) before sequencing.
How long can I store my MagBead bound sample?
- PacBio recommends that MagBead samples be stored at 4C in the dark and sequenced as soon as possible.
My MagBeads were accidentally left at room temperature for several hours. Can they still be used?
- In most cases, MagBeads should still be useable by first chilling them at 4C before use.
My MagBeads / AMPure beads were accidentally stored at -20C. Is it still okay to use the beads?
- PacBio does not recommend using AMPure PB beads or MagBeads that have been accidentally stored at -20C because the beads may
become damaged and may leach after being frozen. However, Customers may use them at their own risk after bringing the MagBeads to
4C and AMPure PB beads to room temperature.
When preparing >30 kb SMRTbell libraries, can (AMPure-purified and concentrated) sheared gDNA be stored at 4C for longer than 24
hours?
- PacBio generally recommends that AMPure-purified and concentrated sheared gDNA be stored for up to 24 hours at 4C or at -20C for
longer durations. However, if the gDNA is relatively pure (i.e., free of endonucleases), it should be acceptable to store the sheared gDNA
sample for 2-3 days at 4C.
Conditions for shearing gDNA to a size that can support producing ≥30 kb libraries must be determined and verified empirically for
each sample. When preparing ≥30 kb SMRTbell libraries using Megaruptor, what is the recommended target shear size if the desired
size selection lower cutoff is, for example, 15-20 kb, 30 kb, or 40 kb?
- When preparing ≥30 kb SMRTbell libraries using Megaruptor, the recommended target shear size depends on the size selection lower cutoff
to be employed. The Table below may be considered a useful starting point; but empirical optimization and accurate size quantitation are
essential:
Library
Insert Size
(kb)
Size Selection
Lower Cut (kb)
Target gDNA
Shear Size
(kb)
30 15 - 20 30
30 - 40 15 - 20 50
40 - 50 30 60
50 - 60 40 75
Where can I find the Plate Map and sequences of all the primers in the Barcoded Universal F/R Primers Plate - 96 (P/N 100-466-100)
product and Barcoded Adapter Plate - 96 (P/N 100-466-000) product?
- To obtain the sequences of the primers used in the Barcoded Universal F/R Primers Plate - 96 Kit, please contact your local Field
Applications Scientist, or submit your inquiry through the PacBio Customer Portal (http://www.pacbioportal.com/) or email
- The Barcode Plate Map Diagram can be downloaded from PacBio’s Documentation webpage (http://www.pacb.com/support/documentation/)
here: http://www.pacb.com/wp-content/uploads/2015/09/User-Bulletin-Barcode-Plate-Mapping.pdf
There is a ‘Barcoding - RSII and SMRT Analysis 2.3.0 or older’ webpage on GitHub
(https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Barcoding). Where can I find the latest guidance on PacBio
Barcoding recommendations for multiplexed sample preparation for Sequel System / SMRT Link v4.0 (or later)?
- The most up to date information on PacBio multiplexing applicable to SMRT Link v4.0 (or later) can be found here:
https://github.com/PacificBiosciences/SMRT-Link/wiki/SMRT-Analysis-Barcoding-Primer
Can I use Illumina 8-bp barcode index sequences for preparing multiplexed samples for PacBio sequencing?
- No; PacBio does not recommend using Illumina 8-bp barcode index sequences for preparing multiplexed samples for PacBio SMRT
sequencing applications.
How are the 16-bp PacBio barcodes incorporated into the SMRTbell DNA template?
- PacBio uses two approaches:
- Adding a barcode to end of the standard SMRTbell adapter. The combined adapter is called a Barcoded Adapter.
- Adding a barcode to the PCR amplicon. This approach involves a two-step PCR reaction workflow. The internal primers for the first
PCR are augmented at the 5’ end by universal sequences to the target-specific primers. The external primers contain the 16bp barcode
at the 5’ end and the universal sequences. This approach is called Barcoded Universal Primers.
What are the supported applications for using PacBio Barcoded Adapters and PacBio Barcoded Universal Primers with multiplexed
samples? What are not supported applications?
- Supported applications are sequencing of one species per sample or loci. Examples of supported applications include: Confirmation of
SNPs, resequencing, most Long Amplicon Analysis (LAA) applications, and Sanger sequencing replacement. An exception is HLA typing,
which may have 2 species per loci. Multiplexing of HLA has also been demonstrated with the use of additional custom analyses (see
PacBio’s AGBT 2015 Poster:
http://files.pacb.com/pdf/Poster_MultiplexingHumanHLAGenotyping_DNABarcodeAdapters_HighThroughputResearch.pdf)
- Note: The product specifications for the PacBio Barcoded Adapter Kit and PacBio Barcoded Universal Primer Kit are such that the level of
barcode oligo contamination in the 96-plate wells should not exceed 5%. Therefore it is possible, though unlikely, to have 1 other
contaminant barcode primer/adapter sequence present at levels up to 5%. PacBio does not recommend using the PacBio Barcoded Adapter
Kit and PacBio Barcoded Universal Primer Kit for minor variant detection < 10%.
Does PacBio have any specific DNA polymerase enzyme or Kit recommendations for long-range PCR (LR PCR) for generating long
DNA amplicon samples for sequencing?
- While PacBio does not recommend a specific enzyme, a high-fidelity enzyme is generally preferred. For example, PrimeStart GXL from
Takara and ThermoFisher Phusion Hot Start II DNA Polymerase have given good results to our internal scientists.
- Do PacBio’s target enrichment sample prep protocols/tools serve you well for
your project needs?
-What other things would you like PacBio to add to our current solutions for
targeted sequencing?
-What are your opinions on the current state of SMRT Sequencing for targeted
applications?
Other Discussion Points
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