Misc study deck Flashcards

Question

How does FlexPrep work?

Answer 1

1. Normalization by ligation: Automatically balances samples 2. Early pooling- Mix 12 samples per reaction with UDI's (saves steps) 3. High throughput- 96-well plate, 96 samples per reaction, 4-5 hours total*

Answer 2

1. Agrigenomics: Genotyping cows, plants. Replaces microarrays 2. Human population Genomics: Think big biobank projects like Galatea and trying to sequence 10M genomes.

Answer 3

1. Saves time: skips normalization. 4-5 hours total for prep. No upfront measuring 2. Handles big batches: 1,152 samples in one 96-well plate, ultrahigh throughput 3. Cuts cost- 12x less consumables 4. Beats microarrays- NGS gives more data (beyond SNP's), customizable, non-species specific 5. End to end support- pairs with Gencove for data analysis and Curio for Agrigenomics.

Answer 4

Flexprep preps 1,152 samples in 4 hours. Big batches, big wins.

Answer 5

1. Are you working on agrigenomics or population genomics projects? 2. How many samples are you aiming to run- small batches or millions? 3. Are you using microarrays now? do you want more data? (learn how to sell against arrays)

Answer 6

1. Library Prep: Speed and sample flexibility (EF 2.0 for FFPE and WGS) 2. UDI/UMIs: For scale think UDI and for cancer detection think UMI's 3. RNA-SEq: FFPE and fustion detection (RNA Exome) 4. Methylation: Early cancer detection, beats arrays and spots early signals at 0% 5. FlexPrep: Big batches, cost savings- handles 1,152 samples in 4 hours.

Answer 7

Amplicon Seq vs. PCR in NGS PCR (Polymerase Chain Reaction): PCR is a foundational technique used to amplify specific DNA segments. In NGS workflows, PCR is a preparatory step to generate enough DNA for sequencing. It uses primers, a polymerase enzyme (e.g., Taq), and thermal cycling to exponentially replicate a target region. For example, PCR might amplify a gene of interest to create sufficient material for library preparation before sequencing on an Illumina platform. Role in NGS: PCR is often used in library preparation (e.g., to add adapters or barcodes) or to amplify amplicons for Amplicon Seq. Twist’s 96-Plex Library Prep Kit, for instance, leverages PCR to prepare up to 960 samples at $10/sample, ensuring enough material for sequencing. Amplicon Sequencing (Amplicon Seq): Amplicon Seq is a targeted NGS method where specific DNA regions (amplicons) are first amplified via PCR, then sequenced. It focuses on sequencing these pre-amplified regions, often for applications like microbial diversity (e.g., 16S rRNA sequencing), SNP detection, or variant analysis in specific genes. The process involves designing primers for target regions, amplifying them with PCR, and sequencing the resulting amplicons. Role in NGS: Amplicon Seq is the full workflow—amplification (via PCR) followed by sequencing and analysis. Twist’s Custom Panels (80-90% on-target, 4-week delivery) are ideal for Amplicon Seq, enabling labs to target specific genes with high accuracy. Key Differences Scope: PCR is a single step (amplification) within many workflows, including Amplicon Seq. Amplicon Seq is the broader process, encompassing PCR, sequencing, and analysis. Purpose: PCR generates DNA material; Amplicon Seq delivers sequence data for specific targets. Applications: PCR is used in diverse contexts (e.g., cloning, qPCR, library prep). Amplicon Seq is specialized for targeted sequencing, like studying microbial communities or cancer mutations. Scale: PCR can be low-throughput (e.g., a few reactions). Amplicon Seq leverages NGS for high-throughput sequencing of many amplicons simultaneously.

Answer 8

adapters and polymerase

Answer 9

The Twist Bioscience ligase refers to a proprietary enzyme developed by Twist Bioscience to enhance their Next-Generation Sequencing (NGS) workflows, particularly in their cfDNA (cell-free DNA) Library Preparation Kit. This ligase is a key component of an optimized protocol and buffer formulation designed to improve the efficiency of library preparation for liquid biopsy applications, such as cancer detection. It enables increased conversion efficiency—reportedly doubling the conversion rate compared to other kits—allowing for higher yields, better mean target coverage, and more sensitive detection of low-frequency variants, even with low input amounts (1 to 20 ng/ml). The enzyme also supports fast reaction times, which is critical for handling degraded or low-concentration cfDNA samples often encountered in NGS. This ligase is part of Twist’s broader strategy to refine NGS processes, as seen in their partnerships (e.g., with Watchmaker Genomics for high-fidelity enzymes) and their focus on innovative solutions like the FlexPrep UHT Kit. While the exact composition of the ligase isn’t publicly detailed, its development reflects Twist’s expertise in enzyme engineering to address challenges in precision oncology, such as early cancer detection and tumor profiling. For your prospecting in Miami and beyond, this ligase could be a selling point for labs like Orlando Health, where sensitive cfDNA sequencing could enhance their Cancer Genetics and High Risk Center’s capabilities. Would you like to incorporate this into a pitch for one of your upcoming meetings?

Answer 10

increasing their library complexity. Cost savings.

Answer 11

RAMseq vs. Amplicon Seq vs. PCR PCR (Polymerase Chain Reaction): PCR is a foundational lab technique to amplify specific DNA segments. It uses primers, a thermostable DNA polymerase (like Taq), and thermal cycling (denaturation, annealing, extension) to create millions of copies of a target DNA region. In NGS workflows, PCR is a preparatory step, often used to amplify regions of interest or add adapters during library preparation. For example, Twist’s 96-Plex Library Prep Kit relies on PCR to prepare samples at $10/sample, ensuring enough material for sequencing. PCR is not a sequencing method itself but a critical enabler for methods like Amplicon Seq. Amplicon Sequencing (Amplicon Seq): Amplicon Seq is a targeted NGS method that sequences specific DNA regions (amplicons) after amplifying them via PCR. It involves designing primers to amplify regions of interest, followed by sequencing the amplicons on platforms like Illumina or Ion Torrent. It’s widely used for variant detection (e.g., SNPs, mutations) and applications like microbial diversity (16S rRNA sequencing) or cancer profiling. Twist’s Custom Panels (80-90% on-target, 4-week delivery) are ideal for Amplicon Seq, offering high accuracy for targeted sequencing in labs like Moffitt Cancer Center’s Molecular Genomics Core. RAMseq (Random Amplicon Sequencing): RAMseq is a specialized method for SNP detection in non-model species without a reference genome, using semi-deep sequencing of random amplicons. Unlike Amplicon Seq, which targets specific regions with designed primers, RAMseq amplifies random DNA fragments (often via RAPD—Random Amplified Polymorphic DNA—followed by sequencing). It’s cost-effective for genetic diversity studies in wild species, as demonstrated in a study on the Eurasian otter, where it identified 238 SNPs. However, its random nature makes it less precise for targeted applications compared to Amplicon Seq, and it’s less commonly used in clinical or focused research settings. Key Differences Purpose: PCR: Amplifies DNA as a preparatory step; not a sequencing method. Amplicon Seq: Sequences specific, pre-amplified regions for targeted analysis (e.g., mutations, microbial profiling). RAMseq: Sequences random amplicons for SNP discovery in non-model organisms, focusing on genetic diversity. Targeting: PCR and Amplicon Seq use specific primers for defined regions. RAMseq uses random primers, making it less targeted and more exploratory. Applications: PCR: Foundational for many workflows, including library prep for NGS. Amplicon Seq: Ideal for precision oncology, pathogen detection, and targeted genomics (e.g., at UMMC’s Molecular Genomics Core). RAMseq: Suited for ecological or population genetics studies (e.g., at MSU for sickle cell disease genetics). Scale and Output: PCR: Produces amplicons but doesn’t generate sequence data. Amplicon Seq: High-throughput, yielding detailed sequence data for specific targets. RAMseq: Semi-deep sequencing, yielding broader but less focused SNP data. Relevance to Twist Products Twist Fit for Amplicon Seq: Twist’s portfolio, like the Methylation Detection System (4M CpG sites) or FlexPrep UHT (1,152 samples in 5 hours), aligns perfectly with Amplicon Seq workflows, offering high sensitivity and scalability for labs like NSU’s Genomics Core. Twist Fit for PCR: Twist’s library prep kits (e.g., cfDNA Library Prep Kit with proprietary ligase) optimize PCR steps, improving yield and sensitivity for downstream sequencing, which could appeal to Moffitt’s precision oncology efforts. RAMseq Limitations with Twist: RAMseq’s random approach doesn’t align as directly with Twist’s targeted NGS tools, though its library prep kits could still support initial amplification steps for labs like MSU exploring genetic diversity.

Misc study deck Flashcards

(35 cards)