HTG EdgeSeq System Workflow Overview

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1 HTG EdgeSeq System Workflow Overview

2 The HTG EdgeSeq system is an automated chemistry and workflow solution based on HTG Molecular Diagnostics (HTG) extraction-free sample preparation technology and quantitative nuclease protection chemistry (qnpa). This assay is used with the next-generation sequencing (NGS) technology to produce quantitative expression profiles. Sample-to-answer in about 36 hours Leveraging extraction-free chemistry and automated nuclease protection, the HTG EdgeSeq chemistry and workflow combined with next-generation sequencing provides digital gene expression profiles in about 36 hours. Sample Prep The HTG EdgeSeq system overcomes a key sample bottleneck for expression analysis by using extraction-free, lysis-only chemistry to significantly reduce sample input requirements from limited, precious FFPE tissues, plasma, serum, whole blood and cells. For example, for most HTG EdgeSeq assays, mm 2 of a single 5 µm FFPE tissue section is all that is needed for expression profiling. Sample lysates are dispensed into the 96 well plate provided with each HTG EdgeSeq assay and transferred to the HTG EdgeSeq processor where the nuclease protection step in the library preparation process is fully automated, thus significantly reducing the number of handson steps. Library Preparation/Quantitation Processed samples undergo library preparation (target protection and library amplification), pooling, cleanup and QC followed by sequencing on either the MiSeq or Ion Torrent sequencers. Sequencing & Data assembly After sequencing is complete, the FASTQ files are loaded into the HTG EdgeSeq parser software which is included with the system. Data output takes approximately minutes.

3 Sample Prep Simplified workflow supporting many sample types FFPE Tissue Plasma/Serum PAXgene Cell Lines Purified RNA The HTG EdgeSeq assays are compatible with multiple sample types (such as FFPE, plasma/serum, PAXgene, cells, and purified RNA). Samples are first lysed using our proprietary lysis buffers, depending on the sample type and then loaded into the HTG EdgeSeq system for library preparation. HTG EdgeSeq assays come with everything that is needed to process the most common sample sources. Users are supplied with HTG EdgeSeq lysis buffer, biofluids and plasma lysis buffers, along with Proteinase K and denaturation oil. Sample lysates are transferred directly into the HTG EdgeSeq automated platform. Minimal sample manipulation 30 mins hands-on time Automated processing C US For most sample types, the lysis step requires minimal hands-on time and takes approximately 30 minutes. Batch times and total sample preparation time may vary depending on the number of sample types and quantity planned for testing.

4 Library Preparation/Quantitation Minimal enzymatic processing Take advantage of extraction-free, nuclease protection chemistry HTG EdgeSeq assays employ proprietary qnpa to capture expressed transcripts. Target-specific protection probes with universal priming sites (wings) bind to target transcripts and wingman (1). The probe, transcript and two wingman molecules together produce a heteroduplex. S1 nuclease is added to degrade the non-hybridized probes and non-targeted RNA, resulting in a 1:1 stoichiometric ratio of DNA probes to the targeted RNA (2). After S1 inactivation step and elimination of the RNA with heat and base, the protection probe is ready for tagging (3). Sequencing adaptors and molecular barcodes are then added to the remaining probes using a simple PCR reaction (4). The resulting PCR products are then cleaned and quantitated and are ready for subsequent sequencing (5). HTG EdgeSeq assays do not require processing steps such as reverse transcription, adenylation or ligation

5 Sequencing & Data Assembly Configurations for multiple sequencers Illumina HTG EdgeSeq assays are compatible with common next-generation sequencing instruments. Because sequencers requirements vary, certain HTG EdgeSeq assays may not have a configuration for your sequencer. Refer to the latest documentation specific to your assay to determine if a configuration exists with your platform. Ensure success using common lab reagents and workflows HTG EdgeSeq assays require the following reagents purchased from third-party vendors: NEB OneTaq PCR Master Mix or NEB Hemo Klentaq PCR enzyme + NEB OneTaq GC Reaction Buffer Agencourt AMPure XP beads for library cleanup Kapa Quant-It qpcr assay for library functional assessment and quantification. Alternatively, labs may use their preferred method of QC prior to loading Faster quantitative results with HTG EdgeSeq parser software Thermo Fisher HTG EdgeSeq system comes pre-installed with the HTG EdgeSeq parser software. This software uses FASTQ files and the sample sheet generated by the HTG EdgeSeq host software to generate quantitative expression profiles. The process takes as little as 15 minutes and requires no complex bioinformatics analysis to complete. Sample FASTQ File Sample Sheet Parsed Data

6 Performance Maximize data from precious samples Because the HTG EdgeSeq chemistry does not require extraction, thus avoiding the yield losses associated with extraction, researchers can achieve maximum performance from small sample input quantities. The minimum sample requirements recommended by HTG allow for retention of precious samples and enables repeat testing of the same sample to increase the confidence in the expression results. Scatterplot Matrixs Sample input for HTG EdgeSeq mirna Whole Transcriptome Assay Sample Type Requirements FFPE mm 2 of a 5 µm section Cell Lines 1,250-5,000 cells Plasma/Serum 12.5 µl PAXgene 12.5 µl Purified RNA ng The HTG EdgeSeq mirna Whole Transcriptome Assay with probes targeting 2,083 human micrornas produces consistent results in titrations from 1,250-5,000 cells (H520) (right) and from mm 2 ovary tumor surface area from a single 5 µm FFPE slide (left). Scatterplot Matrix 10 r= r= Excellent cross-platform performance The HTG EdgeSeq chemistry demonstrates consistent results across different sequencing platforms. In the experiment shown to the left a FFPE tissue sample was lysed and processed using HTG EdgeSeq sample prep chemistry. From that single lysed sample, libraries were prepared in parallel for the Ion Torrent PGM and Illumina MiSeq instruments using HTG EdgeSeq IT sequencing tags and HTG EdgeSeq ILM sequencing tags, respectively. The Ion Torrent PGM library was sequenced by a third party, academic genomic services lab, while an Illumina library was sequenced at HTG using the Illumina MiSeq instrument. FASTQ data from both instruments were aligned and mirna expression was reported using the HTG EdgeSeq parser software. The data was analyzed using JMP statistical analysis software.

7 HTG EdgeSeq assays for NGS-based expression profiling mirna Whole Transcriptome Assay 2,083 human mirna transcripts Immuno-Oncology Assay 549 key tumor immune response genes Oncology Biomarker Panel 2,560 key tumor biomarkers Lymphoma Panel 93 lymphoma associated genes DLBCL Cell of Origin Assay RUO Subtype DLBCL tumors, lineage markers HTG EdgeSeq system highlights Test Type Specimen Types Multiplexing Turn-around time Plate type System modularity Detection system mrna, mirna Cell lines, fixed tissue, plasma, serum, PAXgene, and purified RNA Up to 2,560 targets per well About 24 hours sample and library prep 96 well HTG barcoded microtiter plate Up to 5 processors per host PC Next-generation sequencing Ordering information or contact your local sales representative. C US General Product # Configuration Processor weight Processor size Operating environment Input Noise specifications Warranty HTG Edge-SQ-100 (processor and host PC) Benchtop 225 lbs (102 kg) 17.3 in x 36.0 in x 24.0 in (43.9 cm x 91.4 cm x 61.0 cm) Temperature 15 C 30 C (59 F 86 F) VAC, Hz, 6 Amps VAC, Hz, 6 Amps < 65 dba 1 year

8 HTG Molecular Diagnostics, Inc E. Global Loop, Tucson, AZ Call Website For Research Use Only. Not for use in diagnostic procedures. HTG EdgeSeq, HTG Edge and qnpa are trademarks of HTG Molecular Diagnostics, Inc. Any other trademarks or trade names used herein are the intellectual property of their respective owners. Revision 3: 9-JUN-2016

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