How the products work in the lab Flashcards
Step 1
Sample comes in. DNA or RNA maybe from a tumor biopsy (FFPE), blood (cfDNA) or a plant leaf. it’s usually a mess. low amounts, degraded or mixed in with a lot of “junk”.
Step 2
Library Prep- with the Twist library prep kits, they take the raw sample and turn it into a “library” which is esentially a collection of DNA/RNA fragments tagged and ready to hit the sequencer.
Ex: Twist RNA library prep kit. You toss in 1ng of FFPE RNA, enzymes chop it up, adapters get slapped on and in 5 hours, you have a library with unique bar codes (UDI adapters) so you can track it.
Step 3
Target enrichment- Twist probes are the star of the show here, they swoop in and grab only the bits you care about (exons, RNA, transcripts)
Ex: Twist human core exome kit.
How it works- double stranded probes (made on the silicon chip) hybridize to your library, fishing out the exome (protein-coding regions). magnetic beads pull the caught material and the rest gets washed away.
Step 4
Reagents- Twist’s reagents like the fast hyb mix or rRNA depletion kits juice up the process.
Ex- rRNA and globin depletion kit
How it works- add this to the RNA library and it zaps out ribosomal RNA “noise” from blood samples in 2 hours, leaving clean data behind.
Step 5
The sequencer- load the enriched library onto the sequencer (NovaSeq) and because of Twists uniformity, you will get deep, even coverage without overspending on reads.
Real world example- Cancer research and FFPE tumor samples
Goal- find mutations in a lung cancer patients tumor sample.
Sample- FFPE tissue from a biopsy, low RNA and very degraded.
Twist tools:
- RNA library prep kit: Turns 10ng of RNA into a library in 5 hours.
-RNA exome probes: sangs coding transcripts (exons) with an “exon aware” design, even from the trash sample.
-rRNA depletion reagent: clears out ribosomal “junk”
Lab flow steps: Extract the RNA, prep library, enrich with probes, sequence on Illumina and find a rare gene fusion that’s driving the cancer.
