Workflows and applications
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The STM Genomics platform at the Alfred Campus combines the latest technologies with a diverse range of workflows to deliver first-class genomic data at a large scale. By utilising high-throughput sequencers, researchers are encouraged to conduct sequencing experiments of varying complexity and capacity. With expert support available on-site, STM Genomics is here to provide experience and knowledge for researchers looking to take on high-impact projects.
Custom sequencing conditions are available (ie. custom primers, sequencing cycle conditions). Conditions apply. Please contact STM Genomics to discuss options.
Sequencing Instruments Available
Instrument Reagent Kit Read Output 100 cycle PE50 150 cycle PE75 200 cycle PE100 300 cycle PE150 500 cycle PE250 600 cycle PE300
NovaSeq 6000SP 650-800M 65–80Gb 134–167Gb 200–250Gb 325-400Gb S1 1.3-1.6B 134–167Gb 266–333Gb 400–500Gb S2 3.3-4B 333–417Gb 667–833Gb 1000–1250Gb S4 8-10B 1600–2000Gb 2400–3000Gb
NextSeq 500Mid-Output 130M 16.2–19.5Gb 39Gb High-Output 400M 50–60 Gb 100–120Gb
MiSeqMicro kit v2 4M 1.2Gb Reagent kit v2 12-15M 4.5–5.1Gb 7.5–8.5Gb Reagent kit v3 22-25M 3.3–3.8 Gb 13.2–15Gb
MiniSeqMid-Output 7–8M 2.1–2.4Gb High-Output 22–25M 3.3–3.7 Gb 6.6–7.5Gb - PE - Paired end
- M - Million
- B - Billion
- Gb - Gigabases
The NovaSeq 6000 flow cell can be divided into lanes using the XP workflow. Each lane can be used separately, allowing for projects that do not require the full flow cell. The SP, S1, and S2 flow cells have two lanes while the S4 has 4.
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Sample Quality
Agilent TapeStation 4200
QC analysis for:
- Extracted samples – RNA and DNA tapes
- The TapeStation 4200 system separates nucleic acids by means of electrophoresis. The system detects:
- Double stranded DNA including genomic and cell-free DNA
- Total RNA (Eukaryotic and Prokaryotic)
- The TapeStation 4200 system separates nucleic acids by means of electrophoresis. The system detects:
- Prepared Libraries – D1000 and D5000 tapes
- Prior to assembling libraries for sequencing, purified DNA and RNA samples should be checked for quantity, purity, and integrity (RIN for RNA or DIN for DNA), to save time and avoid problems downstream in the workflow.
- Libraries prepared for sequencing also need to be tested for contaminants and impurities, like primer and adapter dimers, which can affect binding metrics and efficiency. For this purpose, all libraries submitted to STM Genomics for sequencing undergo QC testing on the TapeStation 4200 as it allows us
Sample Quantification
Qubit 3.0
- DNA Assays
- Broad Range (BR) dsDNA : 0.2–4,000 ng/µl
- High Sensitivity (HS) dsDNA: 0.005–120 ng/µl
- ssDNA: 0.05–0.2 ng/µL
- RNA Assays
- Broad range (BR): 1–1000 ng/µl
- High Sensitivity (HS): 0.25–100 ng/µl
- Extracted samples – RNA and DNA tapes
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Bio-Rad QX200 Droplet Digital PCR (ddPCR) System
The QX200 Droplet Digital PCR (ddPCR) System (https://www.bio-rad.com/en-au/life-science/digital-pcr/qx200-droplet-digital-pcr-system) by Bio-Rad is designed for absolute quantification of nucleic acids with exceptional precision. Unlike traditional quantitative PCR (qPCR) methods that rely on standard curves, ddPCR partitions each sample into approximately 20,000 droplets, each containing an individual PCR reaction. This partitioning provides more accurate detection and quantification of low-abundance RNA molecules, including those exhibiting small fold changes.
Bio-Rad’s ddPCR workflow utilises TaqMan hydrolysis probes labelled with FAM and HEX (or VIC) fluorophores to detect the presence or absence of the target sequence in each droplet. After amplification, the system applies Poisson statistics to calculate the absolute number of target molecules in the original sample, eliminating the need for reference standards or calibration curves.
Notable Applications
- Absolute Quantification: Direct measurement of nucleic acid concentration without standard curves.
- Copy Number Variation (CNV) Analysis: Accurate determination of gene copy numbers.
- Rare Mutation and Sequence Detection: Identification of low-frequency variants in mixed populations.
- Gene Expression: Reliable and reproducible measurement of small fold changes in low-abundance mRNA and miRNA.
Key Advantages
- Unparalleled Precision: Sample partitioning delivers the capability to detect genes exhibiting small fold changes.
- Increased Signal-to-Noise: Enriches rare targets by reducing competition from high-copy templates.
- Elimination of PCR Efficiency Bias: Reduces error rates by removing reliance on amplification efficiency, enabling accurate quantification.
- Simplified Quantification: No standard curve or technical replicates are required for absolute quantification.
The Bio-Rad QX200 Droplet Digital PCR system is operated by STM Genomics, and assays are processed on a per-sample basis. Hydrolysis probes required for the assays should be submitted along with samples. For enquiries about sample requirements, pricing, and quotes, please reach out to STM Genomics directly.
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Library Preparation for Next-Generation Sequencing (NGS)
Library preparation is a critical step that enables raw nucleic acids to be sequenced on Illumina instruments and is required for all applications such as RNA sequencing (RNA-seq), whole-genome sequencing (WGS), and targeted DNA sequencing.
How It Works
Illumina library preparation involves fragmenting DNA or RNA and ligating platform-specific adapters to both ends of each fragment. These adapters contain sequences that are complementary to oligonucleotides on the surface of a flow cell allowing library fragments to bind and be sequenced. For RNA-seq, the workflow typically includes mRNA enrichment or rRNA depletion, followed by reverse transcription to generate cDNA. DNA-based workflows, such as WGS or targeted sequencing, often include fragmentation, end repair, A-tailing, and adapter ligation steps.
In addition to adapter ligation, indexes of 6-10bp are added to each library, acting as a barcode for multiplexing within a single sequencing run. After sequencing, bioinformatic tools demultiplex the raw data, assigning reads to their corresponding sample.
RNA Sequencing (RNA-seq)
- Whole Transcriptome RNA-seq: Captures all RNA species, including coding and non-coding transcripts, for comprehensive transcriptome profiling. Ideal for studies of gene regulation, alternative splicing, and novel transcript discovery.
- Poly-A mRNA-seq: Enriches for polyadenylated RNA species (mRNA), focusing on protein-coding transcripts. Commonly used for differential gene expression analysis and functional genomics.
- Ribosomal RNA-Depleted RNA-seq: Removes rRNA to enable sequencing of both coding and non-coding RNAs, including lncRNAs and other regulatory RNAs. Useful for exploring transcript diversity beyond mRNA.
- Small RNA-seq: Targets short RNA species such as microRNAs (miRNAs) and siRNAs, providing insights into post-transcriptional regulation and small RNA-mediated pathways.
DNA Sequencing (WGS and Targeted Approaches)
Illumina DNA-seq workflows cover a broad range of applications, from whole-genome sequencing to highly targeted panels:
- Whole-Genome Sequencing (WGS): Provides complete genomic coverage for variant discovery, structural variation analysis, and de novo assembly.
- Targeted Gene Panels: Enables deep sequencing of specific genes or regions for cancer genomics, inherited disease studies, and pharmacogenomics.
- Amplicon Sequencing: PCR-based enrichment for high-sensitivity detection of mutations or microbial targets.
- Metagenomics: Profiles microbial communities and functional gene content from environmental or clinical samples.
Epigenomics and Chromatin Studies:
Library preparation workflows can be adapted for epigenetic profiling:
- DNA Methylation Analysis: Analyse the 5-mC and 5-hmC methylation signatures within CpG-rich regions using Enzymatic Methyl-seq (EM-seq).
- ATAC-seq: Assess chromatin accessibility by using the Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin.
- ChIP-seq: Map protein-DNA interactions for transcription factor binding sites and histone modifications.
STM Genomics provides a range of library preparation options for sequencing on Illumina instruments. Workflows requiring a tailored approach are dependent on feasibility, and we will work with you to find the most suitable solution. For more information, please submit an enquiry.