~Library preparation for metabarcoding~
Sorting
- The first step of sample processing that occurs in the lab is sorting. This can involve identifying, weighing, size sorting, and/or taking photos of the arthropods. This information is important for later analysis alongside genomic data. For our project, we record the wet weight of the sample and take photo vouchers to be referred to after DNA extraction and analysis.
DNA extraction
- Lyse cells
- Extract DNA
- Isolate DNA
- Result: accessible and isolated DNA
TS PCR
- Amplify targeted sequences of extracted DNA
- Result: many copies of specific DNA fragments
Gel Electrophoresis
- TS PCR product is mixed with dyes and loaded into wells at one end of the gel in a running buffer
- An electric current across the matrix causes DNA fragments to migrate towards the positive node and separate by size
- DNA is negatively charged due to its phosphate groups so it will migrate towards a positively charged node
- smaller fragments move farther/faster; larger fragment move slower/less far
- Negative control and ladder are run alongside samples to check for contamination and calibration, respectively
- Visualize gel with UV light
- band strength is an indicator for sample concentration
- Result: verification of PCR amplification success and no contamination
- See How to Read a Gel Image
Pooling
- Used unique primers and inline barcodes (ARF1 A/B/C and MCO A/B/C) in the first round of PCR so can pool samples together
- Pooling based on relative band strength (from gel image), which indicates concentration
- Goal: combine samples so there is relatively equal molar amounts of TS-PCR product combined togetherResult: samples ready for Index PCR
Index PCR
- Second round of PCR
- Allows multiple libraries to be pooled and sequenced together
- Great for metabarcoding bulk arthropod samples