Back to basics - the crucial step of sample collection

Posted on Wed, Jan 15, 2020 @ 09:37 AM

Best practice in sample collection for SeqSNP targeted genotyping by sequencing projects

When good goes in, great comes out

When good goes in, great comes outGreat genotyping data begins with good sample collection. Sampling can be tedious work, so we’re providing tips on proper collection to lock down efficiency and ensure the best outcome.


When your sample number is getting into the thousands, make sure you’ve planned ahead to accommodate for:

  1. Training staff on how to sample consistently in both the stage of growth and number of samples needed, e.g. leaf punches.
  2. The amount of time it will take to sample.
  3. When and where the samples will be extracted.

Giving yourself ample time to properly collect your samples will reduce your stress, maximise on efficiency, and safeguard yourself from wasting any time or effort if done correctly. That way, you’re setting yourself up for success to produce quality data down the road. 

For SeqSNP projects, the minimum number of samples that can be submitted is 48; however, we recommend a minimum of 96 samples due to the scales of the kits. Samples must be submitted in 96-well plates, see plating instructions below. Any samples submitted in tubes may incur a surcharge for sample transfer (excludes blood or buffy coat samples).

Accepted formats include:

1. Extracted DNA may be shipped on ice or at room temperature. The key here is that you need enough of it with sufficient quality and purity to produce the best data.

Sample requirements

Plating instructions
  1. Minimum of 20 µL of DNA per individual sample at minimum concentration of 30 ng/µL
  2. DNA must be submitted in solution
  3. High molecular weight DNA (average fragment length >10 kb)
  4. RNA-free
  5. Eluted in Tris/TE (5mM, pH: 8.5)
  1. No requirement for any empty wells as controls
  2. Samples may be formatted in rows or columns 
  3. Each plate should be labelled and matched to the corresponding sample information in the sample submission form

 

Ensure the sampled material is of the same age across your sampling population. This is because the rate of cell division varies greatly in differently aged plants, which therefore affects the amount of DNA that can be extracted. Older leaf tissue may require more leaf punches (e.g. 7-9) vs. young leaf tissue which may require less (e.g. 4-6) in order to provide sufficient DNA.

2. Plant samples
Here are some guidelines for plant sampling:

  • Sample from consistent positions of each plant.
  • Avoid midrib and axillary veins on leaves.
  • Sample leaf material at L3/L4 stage (i.e. 2-3 leaves on the main shoot, and third/fourth leaf has appeared).
  • Considerations: As DNA extraction efficiency is highly crop and sample-type dependent, it is possible that our extraction protocol may require optimisation to ensure sufficient DNA is obtained. This may involve a short pilot study of your species. Contact us to learn more.
  • If submitting seeds, ensure they are stored dry and ungerminated and that they fit easily into the BioArk Seed Collection Kit for submission.

Check to see if your plant species is listed on our current plant species list.

If yes: If no:
  • Submit samples via the BioArk Leaf Collection Kit
    • For BioArk kits, always re-package the plate in its corresponding box as they have matching barcodes.
    • Use a fresh leaf punch and mat for each collection kit.
    • Avoid puncturing the plastic bag when inserting the completed kit into the bag.
    • Do not reopen the plastic bag once the completed sample kit is contained. 
    • Store completed kits in the dark at room temperature.
  • 6-8 leaf discs per sample is required, although the number of discs required to yield sufficient DNA is highly dependent on the age of the leaf sample and genome size.
  • For seed samples, use BioArk Seed Collection kits to improve organisation, storage, and shipping efficiency while avoiding samples transfer fees and delays in the extraction facility. 
  • Submit DNA extracted as per requirements above, preferably in BioArk kits.
  • Discuss a pilot extraction project with our specialists before commencing.
  • Note that protocol development is not included in SeqSNP service project price and standard turnaround times and conditions may not be achievable with custom protocols. 

Our BioArk Collection Kits improve the quality of the DNA that is extracted from the plant material which in turn yields dramatically improved genotyping data (Figure 1).

Figure 1

 DNA quality without using the BioArk Leaf*    KASP™ genotyping data with BioArk Leaf**

  Data quality without using the plant kit                           KASP genotyping data with the plant kit   

*Genotyping data from plant leaf samples collected without using BioArk Leaf and extracted using non-LGC, Biosearch Technologies extraction chemistry. The call rate is 71.1%.

**Genotyping data from plant leaf samples collected with the BioArk Leaf and extracted in our service laboratory using sbeadex™ extraction chemistry. The call rate is 99.5%.

 

3. Non-plant tissue samples, e.g. whole blood, buffy coat or solid tissue
  • We pride ourselves in collaborating with our 
    customers to create custom solutions to meet their specifications. Please enquire prior to commencing your project.
  • For aquaculture, we have the BioArk Fish Collection Kit to collect regularly-sized fin clips directly into an extraction-capable 96-well format without the use of ethanol, freezing or buffers. This greatly improves storage and shipping logistics and helps protect samples from degradation associated with defrosted packages.

We recommend performing DNA extraction using sbeadex™ chemistry, which delivers nucleic acids of high yield, purity and quality. We conveniently have kits specific for plant and livestock. Check out this application note on seed extraction using sbeadex plant kit. 


Or you can outsource this step to our DNA extraction services team. If you have any questions on sample collection and preparation, our team of experts are happy to help! 

 

Contact technical support

 

Topics: Genomics, Genotyping, Sequencing, SeqSNP

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LGC, Biosearch Technologies is the complete Genomics portfolio from LGC. Providing genomic analysis tools, instrumentation and services to the genomic scientific discovery sector worldwide, with focus on across ag bio, pharma and molecular diagnostics. Visit our home page to view our products and services.

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