Agilent SureSelect Cancer All-In-One NGS Sequencer Product Overview Guide PDF

SureSelect Cancer All-In-One NGS Target Enrichment

Product Overview Guide For Research Use Only. Not for use in diagnostic procedures.

What is All-In-One Target Enrichment? 2

Kit Part Numbers 4

Designing Custom SureSelect Cancer All-In-One Panels 5

Protocol Considerations for the SureSelect Cancer All-In-One Assay 6

Analyzing the Results in SureCall 7

Workflow Guidelines 9

What is All-In-One Target Enrichment?

What is All-In-One Target Enrichment?

Agilents SureSelect Cancer All-In-One is a targeted next-generation sequencing (NGS) solution that enables you to use a single SureSelect assay to interrogate genomic regions of interest for a variety of features including the following:

SNVs (single nucleotide variations) Indels (short insertions and deletions) CNVs (copy number variations) including gene amplifications and deletions Translocations

The targeted NGS libraries are prepared using the SureSelectXT HS or SureSelectXT Low Input Target Enrichment system, using a library of probe oligos that can be designed to capture various types of variants in genes of interest. The resulting enriched NGS libraries are sequenced on the Illumina platform, and then the data are analyzed using Agilents SureCall software.

The oligos in a SureSelect Cancer All-In-One probe library can be designed to target one or more of the following genomic regions for the specific types of variants detailed in Table 1.

Custom SureSelect Cancer All-In-One panels of up to 24 Mb are designed using a dedicated workflow in Agilents SureDesign web application, as detailed on page 5. Pre-designed panels designed to capture variants in genes of interest for lung or solid tumor cancer investigations are also available.

Library preparation and target enrichment using a SureSelect Cancer All-In-One panel is done following the standard SureSelectXT HS system protocol or SureSelectXT Low Input system protocol, using the additional considerations outlined on page 6. Figure 1 shows an overview of the SureSelect Cancer All-In-One NGS sample preparation workflow.

Once NGS data is collected, Agilents SureCall software (v4.1 or later) offers a workflow to analyze the sequencing data from a SureSelect Cancer All-In-One assay. SureCall uses the sequences captured by the variant type-specific probes (see Table 1) for the analysis of the corresponding types of variants. The results are accessible using the Triage View window and the Reports menu. See page 7 for more information.

Table 1 Variants detected using SureSelect Cancer All-In-One assays

Genomic Region Targeted Variant Types Detected

Coding exons and exon-intron boundaries SNVs, Indels

Selected introns Translocations

Genome-wide and gene-regional CNV backbone regions

CNVs (gene amplifications and deletions)

SureSelect Cancer All-In-One NGS Solution 2

What is All-In-One Target Enrichment?

Figure 1. The SureSelect Cancer All-In-One target enrichment workflow.

Capture Library/prepared DNA library hybrids

Captured DNA libraries

Target-enriched DNA library samples ready for sequencing

Hybridize the SureSelect Cancer All-In-One probe library with the prepared DNA libraries

Capture probe library/prepared DNA library hybrids

PCR amplify

Prepared DNA libraries

DNA fragments

Indexed DNA libraries

Covaris Shearing or Enzymatic

Prepare samples using SureSelect XT HS

Library Prep Kit

PCR amplify

Genomic DNA samples ( ng DNA per sample)

SureSelect Workflow

SureSelect Cancer All-In-One NGS Solution 3

Kit Part Numbers

Kit Part Numbers

The SureSelect Cancer All-In-One system is provided as a bundle of a probe capture library with either a SureSelectXT HS reagent kit or a SureSelectXT Low Input reagent kit (see Table 2). Custom SureSelect Cancer All-In-One capture libraries are designed using Agilents SureDesign web application as detailed on page 5. Probe designs for the pre-designed SureSelect Cancer All-In-One Lung and Solid Tumor capture libraries may also be accessed using the SureDesign web application.

Agilents Human Reference DNAs, listed in Table 3 below, can be used as unmatched reference samples for SureSelect Cancer All-In-One analysis, as detailed on page 6.

Table 2 Reagent Kit + Probe Capture Library Part Numbers

Probe Capture Library

SureSelect XT HS Kits SureSelect XT Low Input Kits

16 Reactions, with Index Primers 116

16 Reactions, with Index Primers 1732

96 Reactions, with Index Primers 132*

* Includes 3 single-reaction vials of each index primer 132, for a total of 96 library preparations.

96 Reactions, with Index Primers 196 (manual/automated)

96 Reactions, with Index Primers 97192 (manual/automated)

Custom 1499 kb G9704A G9705A G9706A G9707A/G9507A G9708A/G9508A

Custom 0.5 2.9 Mb G9704B G9705B G9706B G9707B/G9507B G9708B/G9508B

Custom 35.9 Mb G9704C G9705C G9706C G9707C/G9507C G9708C/G9508C

Custom 611.9 Mb G9704D G9705D G9706D G9707D/G9507D G9708D/G9508D

Custom 1224 Mb G9704E G9705E G9706E G9707E/G9507E G9708E/G9508E

Pre-designed Lung G9704R G9705R G9706R G9707R/G9507R G9708R/G9508R

Pre-designed Solid Tumor

G9704S G9705S G9706S G9707S/G9507S G9708S/G9508S

Table 3 Optional Agilent Human Reference DNA Products

Product Part Number

OneSeq Human Reference DNA, Male 5190-8848

OneSeq Human Reference DNA, Female 5190-8850

SureSelect Cancer All-In-One NGS Solution 4

Designing Custom SureSelect Cancer All-In-One Panels

Designing Custom SureSelect Cancer All-In-One Panels

Use the wizard-guided SureSelect All-In-One design workflow in Agilents SureDesign web application to design your SureSelect Cancer All-In-One capture panels (see Figure 2, below). You can create a custom panel by selecting target genes from a list of pre-defined cancer genes and by entering genes or regions of interest to detect specific types of variants. Special considerations for designs that target CNVs and translocations are discussed below.

For any Cancer All-In-One custom panels that target one or more genes for CNV-detection, a small genome-wide CNV backbone of 100 kb probe size is added in the panel design. In addition, regional backbone probes that are evenly spaced along the transcribed regions of the CNV target genes, plus Agilent-defined padding, are also included. These backbones collectively provide copy number normalization, and single-nucleotide polymorphism (SNP) information for Agilents SureCall CNV algorithm to estimate the clonal fraction of each CNV call.

Translocations frequently occur in intronic and intergenic regions that are more likely to contain repetitive sequences. Designs containing translocation hotspot regions can have lower capture specificity than exon-focused designs, leading to a lower percentage of reads on-target. In addition, very small designs (<50 kb) may show a suboptimal percentage of reads on-target. Accordingly, if a custom Cancer All-In-One panel design is less than 50 kb, we recommend that you add additional targets or empirically increase the sequencing depth to compensate for the lower percentage of reads on-target. Agilents Tier 1 custom probe designs can include up to 500 kb of targets.

Your custom panels can be ordered using the Order link associated with each panel design on SureDesign.

Figure 2. The SureSelect All-In-One custom design interface in SureDesign, used to define genes of interest for specific types of variants.

SureSelect Cancer All-In-One NGS Solution 5

Protocol Considerations for the SureSelect Cancer All-In-One Assay

Protocol Considerations for the SureSelect Cancer All-In-One Assay

Perform library preparation and target enrichment using a SureSelect Cancer All-In-One panel according to the instructions in the SureSelectXT HS system protocol or the SureSelectXT Low Input system protocol, using the additional considerations listed below.

Use of a reference sample

In addition to sequence data from the experimental sample, SureSelect Cancer All-In-One system analysis in Agilents SureCall application requires sequence data from a reference sample (either matched or unmatched) without aberrations in the regions of interest. Reference sample characteristics are especially important for CNV detection. For highest CNV-detection sensitivity, include a matched-normal (non-tumorous tissue) reference sample for tumor-normal paired analysis. Otherwise, include a sex-matched or a mismatched reference sample each time you perform target enrichment on a set of experimental samples. Use 50 ng of the reference DNA as the starting material in the protocol.

Agilents Human Reference DNA products are recommended for use as unmatched reference samples (ordered separately, see Table 3 for information).

If you elect to use your own reference sample instead of an Agilent-supplied reference sample, the key requirement is that the sample is diploid and has no or minimal CNV aberrations. Additionally, you must first validate any non-Agilent reference DNA by analyzing the chosen reference sample against a well-characterized sample with known aberrations to make sure that SureCall is calling CNVs and other aberrations accurately when compared to the chosen reference sample.

Recommended sample types, tumor content and DNA input amount

SureSelect Cancer All-In-One system supports the use of DNA samples isolated from cell lines, fresh-frozen tissues, and FFPE tissue blocks. Agilent has not validated the SureSelect Cancer All-In-One system using DNA isolated from ctDNA or needle aspiration samples. Use of tumor samples with at least 15% tumor cell content is recommended.

The optimal range of input genomic DNA is 50200 ng for samples to be enriched and analyzed using the SureSelect Cancer All-In-One system. The DNA input range of 10200 ng listed in the SureSelectXT HS or SureSelectXT Low Input Target Enrichment system user manual can be used, with potentially reduced sensitivity for samples processed using <50 ng DNA.

FFPE sample quantitation

For FFPE samples, Agilent recommends sample qualification using the qPCR-based Agilent NGS FFPE QC Kit, and then using the qPCR-determined concentration of amplifiable DNA when preparing input DNA samples. If qPCR methods are unavailable, it is also acceptable to use quantities of input DNA determined using Qubit assays or using Agilents Genomic DNA ScreenTape assays.

NOTE It is possible to use pre-established reference sample data from a sample that was previously captured using the same probe capture library. However, the potential bias due to batch differences may increase the copy number noise and negatively impact the accuracy of CNV-calling.

SureSelect Cancer All-In-One NGS Solution 6

Protocol Considerations for the SureSelect Cancer All-In-One Assay

Exclusion of molecular barcodes during analysis with SureCall

The All-In-One Analysis workflow in the SureCall software (v4.1) currently does not use molecular barcodes to remove duplicate reads. SureSelect Cancer All-In-One libraries prepared using the SureSelectXT HS or SureSelectXT Low Input Target Enrichment system kits and protocols will, however, contain the degenerate molecular barcodes, and it is possible to design custom analysis pipelines that include molecular barcode-based deduplication.

For standard single-indexed samples analyzed with the SureCall All-In-One Analysis workflow (see page 7 for more information), it is not necessary to collect and analyze i5 molecular barcode reads. If your research design includes sample preparation or analysis methods that deviate from this standard workflow, you should determine whether to collect and analyze i5 index reads. Deviations that require i5 index reads include dual-index based demultiplexing using samples prepared with Agilents Dual Indexing P5 Indexed Adaptors and any custom analysis methods that incorporate i5 molecular barcode reads for single-indexed samples.

Sequencing read length

For translocation detection, Agilent strongly recommends sequencing using at least 2 100 bp and preferably 2 150 bp paired-end reads. For other applications, follow the read length recommendations in the SureSelectXT HS or SureSelectXT Low Input Target Enrichment system user manual.

Sequencing depth

When using 50 ng DNA input, the recommended sequence depth is 4000x or more for 5% variant allele frequency (VAF) and 8000x or more for 1% VAF. Higher sequencing depth may be needed for lower DNA input amounts.

The recommended number of reads for SureSelect Cancer All-In-One Lung and Solid Tumor assays are listed in Table 4 below.

Analyzing the Results in SureCall

Agilents SureCall software v4.1 has the necessary tools and algorithms for analyzing sequencing data from a SureSelect Cancer All-In-One assay. SureCall can perform the analysis using unaligned FASTQ files or aligned BAM files.

The steps for analyzing SureSelect Cancer All-In-One data in SureCall are summarized in Figure 3. The SureCall Help system has specific instructions on each of these steps. Press F1 from any screen in the SureCall software to access the relevant Help topics.

Table 4 Read Number Recommendations

Assay Target VAF Recommended Read Number

SureSelect Cancer All-In-One Lung assay 5% 9 million

1% 18 million

SureSelect Cancer All-In-One Solid Tumor assay 5% 30 million

1% 60 million

SureSelect Cancer All-In-One NGS Solution 7

Protocol Considerations for the SureSelect Cancer All-In-One Assay

Figure 3. Overview of the All-In-One data analysis workflow in SureCall.

SureCall Analysis Workflow for SureSelect Cancer All-In-One Assays

Prepare for sample analysis

Using Matched Reference

Import All-In-One design file from SureDesign

Add unmatched reference file using the Supporting Files tab; review and accept QC data for reference file before use in analysis

On Analysis Workflow tab, from Select Analysis Type menu, choose All-In-One Analysis

Mark the Select unmatched reference(s) checkbox and select the reference file from menu; import sequencing data for experimental sample

Import sequencing data for experimental sample and matched reference sample

On Run Summary screen, review the All-In-One Analysis job parameters then click Run Analysis

Run sample analysis

On Sample Review tab, select sample of interest, and click Triage View to review calls, edit and annotate as desired, and generate reports

View and report results

Enter descriptive information for samples (e.g. sample name, gender, input DNA amount)

Using Unmatched Reference

SureSelect Cancer All-In-One NGS Solution 8

Workflow Guidelines

Workflow Guidelines

Sample Characteristics

Supported Sample Types Sample Details Tumor Content

High-quality DNA samples Fresh-frozen cells, cell lines and other sample types that typically generate high-quality DNA.

15% (measured by Haemotoxylin & Eosin staining)

FFPE DNA samples Obtained from tissue resection (tissue curls or sections on slide). Recommend minimum of 3 sections of 5 m.

15% (measured by Haemotoxylin & Eosin staining)

Unsupported Sample Types Comments

ctDNA SureSelect Cancer All-In-One assays have not been specifically optimized for this sample type, but ctDNA samples may be suitable for SureSelect XT HS or SureSelect XT Low Input library preparation with the modification of omitting DNA fragmentation. For NGS optimization, ensure that the sequencing depth used is appropriate for the expected allele frequency.

Needle aspiration/needle biopsy SureSelect Cancer All-In-One assays have not been specifically optimized for this sample type. If DNA extracted from samples meets the recommended DNA input amount (50 ng), samples may be suitable for SureSelect XT HS or SureSelect XT Low Input library preparation. If the QC analysis of the pre-capture library demonstrates the expected quantity and size profile of library DNA, the libraries may be suitable for use in the SureSelect Cancer All-In-One assay target enrichment and NGS analysis workflow steps.

DNA Extraction and QC

Supported Sample Type

Recommended DNA Extraction Kits

Optimal DNA QC Method and Entry Criteria Alternative DNA QC Method and Entry Criteria

High-quality DNA samples

QIAamp DNA Mini Kit (Qiagen) 50200 ng input DNA, quantified by Qubit BR dsDNA assay (Thermo Fisher Scientific), qualified by AD 260/280

50200 ng input DNA, quantified by Qubit BR dsDNA assay (Thermo Fisher Scientific) or Agilent TapeStation or Bioanalyzer platform assay, qualified by AD 260/280

FFPE DNA samples QIAamp DNA FFPE Tissue Kit (Qiagen) or Agencourt FormaPure Kit (Beckman Coulter)

50200 ng input DNA, qualified by qPCR using Agilent NGS FFPE QC Kit. Recommend Cq 5 for SureSelect Cancer All-In-One assays. Quality results dictate method for quantification: for higher-quality samples with Cq1, use Qubit-determined concentration, for lower-quality samples with Cq>1, use qPCR-determined concentration of amplifiable DNA.

50200 ng input DNA, qualified by Agilent TapeStation Genomic DNA Analysis assay. Recommend DIN 2 for SureSelect Cancer All-In-One assays. Quantify by Qubit BR dsDNA assay.

SureSelect Cancer All-In-One NGS Solution 9

Workflow Guidelines

Fragmentation

Supported Sample Type

Mechanical (Covaris) Shearing Guidelines Enzymatic Fragmentation Guidelines

High-quality DNA samples

Use two-round shearing (2 120 sec) as directed in the SureSelectXT HS system protocol or SureSelectXT

Low Input system protocol.

Use the DNA fragmentation protocol provided in the SureSelect XT HS and XT Low Input Enzymatic Fragmentation protocol. The protocol requires input DNA samples of 50 to 200 ng in 7 l volume. The DNA sample volume may be adjusted by dilution with nuclease-free water or by volume reduction using vacuum concentration, DNA spin cups or other suitable DNA concentration methods.

FFPE DNA samples Use single-round shearing (240 sec) as directed in the SureSelectXT HS system protocol or SureSelectXT

Low Input system protocol. Use the same DNA shearing conditions for all FFPE samples, including highly degraded samples. Mechanical shearing using the Covaris instrument does not further fragment DNA that is already smaller than the target shear size.

Use the same DNA fragmentation conditions for all samples, including highly degraded FFPE samples, to ensure that the DNA fragment ends are suitable for ligation. See the guidelines provided above for high-quality DNA samples.

Pre-Capture QC, Hybridization, and Post-Capture QC Checkpoints

Supported Sample Type

Pre-Capture QC Hybridization Input Post-capture QC

High-quality DNA samples

Quantify and qualify the pre-capture library DNA using Agilent TapeStation or Bioanalyzer system. Electropherograms show peak of DNA fragment size positioned between 300 to 400 bp, with yield typically 500 ng DNA.

5001000 ng prepared library DNA Quantify and qualify the post-capture libraries using Agilent TapeStation or Bioanalyzer system. Electropherograms show peak of DNA fragment size positioned between 200 to 400 bp. Post-capture library yields are highly variable; proceed with sequencing as long as the yield for each sample meets the requirements of your pooling strategy and NGS platform.

FFPE DNA samples Quantify and qualify the pre-capture library DNA using Agilent TapeStation or Bioanalyzer system. Electropherograms show peak of DNA fragment size positioned between 200 to 400 bp, with yield typically 300 ng DNA.

For optimal results hybridize using 500 1000 ng prepared library DNA. In cases where 500 ng of amplified pre-capture library is not available (e.g. for libraries prepared from highly degraded DNA samples), DNA inputs as low as 300 ng may be used, with potential effects on capture performance or NGS metrics.

Sequencing

Supported Sample Type

Library Pooling Guidelines

High-quality DNA samples

Pool equimolar amounts of post-capture libraries to 2 nM, 4 nM, or 10 nM concentration, depending on your Illumina sequencing sample prep protocol. Dry down the pool using vacuum concentration then resuspend at the required volume. Alternatively, you can adjust the sequencing library pool volume using Solid Phase Reversible Immobilization (SPRI) beads.

FFPE DNA samples

SureSelect Cancer All-In-One NGS Solution 10

Agilent Technologies, Inc. 2018, 2019

Revision B0, June 2019

*G9702-90100* G9702-90100

www.agilent.com

In This Book This guide provides overview information for Agilents SureSelect Cancer All-In-One system, including considerations for probe capture library design using Agilents SureDesign application, sample preparation using either SureSelectXT HS

Reagent Kits or SureSelectXT Low Input Reagent Kits, and analysi