Misc study deck Flashcards
What is library prep in NGS?
Getting the samples ready to hit the sequencer. Turning raw DNA/RNA into a sequencing ready library
What is the Twist library prep workflow?
- Fragmentation (chop the DNA/RNA) by mechanical or enzymatic frag.
- size selection- pick pieces (250 bases) -max 600
- End repair/ A-tailing- smooth the ends, add the “A” base for T/A ligation (Twist standard glue)
- Adapter ligation- add barcodes (adapters) to track samples. This uses T4 DNA ligase.
- PCR Amplification- Boost with 3,000+ UDI primers - make enough DNA for sequencing.
Twist EF 2.0 - what does it do? what does it help with?
Enzymatic Fragmentation Kit 2.0- Top seller* combines Fragmentation, End-repair and A-tailing in one step. High throughput and automatic friendly.
Mechanical Frag Kit
Skips fragmentation or shears DNA - best for long-read (PacBrio/ONT) or cfDNA (liquid biopsy)
Selling points for EF 2.0
- saves time- EF 2.0 combines 3 steps into 1. Faster than Illuminas Nextera (which uses tagmentation and gets uneven coverage)
- Handles tough/degraded samples like FFPE
- Scalable- 3,000+UDI primers for massive multiplexing - run tons of samples together
- Cleaner data- avoids tagmentation bias which results in better downstream results.
Customer questions to ask around EF 2.0
- What samples are you currently working with? FFPE, cfDNA or WGS?
(EF 2.0 for FFPE/WGS and mechanical frag for cfDNA) - how many samples are you running at once? (highlight UDI multiplexing power)
- what’s your biggest prep headache- time, bias, sample quality? (EF 2.0 solves time/bias, mechanical frag helps with quality)
What are UDI/UMI adapters?
- Adapters are like labels on your dishes (DNA/RNA fragments) UDI’s track which dish is whose.
UDI (Unique Dual Index)
-10-12 base barcodes for sample tracking.
- Used for multiplexing (running multiple samples together)
- Stops index hopping (misassigning reads)
UMI (Unique Molecular Identifier)
- Tags individual molecules to spot rare variants (cancer mutations)
- Reduces PCR duplicates- noisy copies that mess up data
- Key for MRD and liquid biopsy
UDI= ID
UMI= Unique Molecule
UDI tracks samples; UMI tracks molecules
What are the Twist adapter options?
UDI Adapters- Standard (10 bases) , high throughput (12 bases) -3,000+ options for multiplexing
UMI Adapters- 5-base UMI’s with 2 skip bases, used for low frequency variant detection (cancer)
Full-length UDI- For PCR-free workflows (less bias)
Selling points with our adapters
- massive multiplexing- 3,000+ UDI’s let labs run tons of samples at once. - saves sequencer time.
- Precision for Cancer- UMI’s cut noise. Making rare mutations crystal clear. Hugh in oncology.
- Error-free tracking- UDI’s prevent index hopping- clean sample seperation, even in big runs.
- Flexible applications- UDI’s for standard tracking. UMI’s for precision (think MRD, liquid Bx) and full-length for PCR free.
Customer questions around adapaters
- Are you looking for rare mutations, like in cancer or MRD?
- How many samples do you run together?
- Do you need PCR-free workflows for cleaner data?
Why is RNA data so important?
- shows gene activity (expression levels)
- catches splicing (exon stitching), isoforms, fusions
- Works with FFPE , huge for cancer labs
Twist RNA-Seq workflow
- rRNA depletion- Ribosomal and Globin depletion kit removes rRNA (60-70% noise) and hemoglobin (blood samples) . Does this by our biotinylated probes grabbing the rRNA and the streptavidin beads pulling it out in under 5 hours*
- Less noise= more reads on mRNA, great for FFPE (formalin fixed, perrafin embedded) samples.
- RNA library prep kit (watchmaker sourced) turns RNA into cDNA- strand specific (via uracil integration) : less than 5 hours.
- RNA Exome- targets exons-great for FFPE. detects splicing, known/novel fusions, shadow coverage
- Alliance panels- pre made panels and fusion-focused**
Selling points with RNA Seq
- FFPE Champion- RNA exome works with degraded RNA (1ng) perfect for cancer biopsies.
- Fusion detection- spots known and novel fusions- key for oncology. (leukemia)
- fast workflow- single day prep (less than 5 hours), automation-friendly.
- cleaner data- high on target (80-90%) less sequencing needed. saves costs.
Memory key for RNA Seq
DPT* “Depleat, Prep, Target”
- Remove rRNA
- Make cDNA
- Grab Exons
Customer questions with RNA Seq
- How much FFPE or low quality RNA are you dealing with?
- Are fusions or splicing patterns a priority for you?
- What’s your biggest RNA-Seq headache - time, cost, or rRNA noise?
Methylation
Adds sticky notes (methyl groups) to the DNA cookbook to turn recipes off or down on Cystosine (C) in CpG islands or C/G rich sites
Why does it matter?
- Cancer Detection- hypermethylation (too many sticky notes) or hypomethylation (too few) signals cancer early.
- Drug response- methylation patterns guide drug choice (pharmacogenomics)
- Development/Aging- Big in fetal growth (sperm are hypermethylated) and tracks aging diseases. also think about EpiPaws and how they are using it to help determine age of rescue animals.
Twist Methylation Detection System
- Prep (EM-Seq)
- Capture- human methylation panel covers islands, shores, shelves. Methylation enhancer cuts off-target (20-35%) and also 84% CpG coverage with custom panels available.
Selling points on methylation
- Vs. EPIC array (Illumina 850k) - arrays miss low methylation- Twist detects 0%, covers 4M CpG sites vs. 850k **
- Gentle Sequencing. EM-Seq keeps DNA intact. perfect for liquid biopsies (cfDNA).
- Early detection- spots small methylation changes for cancer.
- cost-saver- high on target (95%), low duplication and less sequencing needed.
- customizable- custom panels for specific genes., fast turnaround.
Memory Aid for methylation
Sticky notes= off
methylation sticky notes turn genes off and Twist finds them.
Methylation customer questions
- Are you studying methylation for cancer or drug response? (helps position panel for pan-cancer or PGx studies)
- Do you work with liquid biopsies or FFPE samples?
- EM-Seq excels with cfDNA/FFPE - How important is detecting low-level methylation changes?
- Twist’s 0% detection beats arrays (5% limit)
FlexPrep UHT
FlexPrep preps 1,152 samples in one go.
