Molecular Minutes

Q&A: Getting Started With Whole Genome Sequencing

Posted by Applied Biological Materials (abm) on November 7, 2019

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Here is the full list of answers to questions we've received during the "Getting Started with Whole Genome Sequencing" webinar: 

    1. How many cells should I use for DNA Isolation for whole genome sequencing?
    2. There is not a reference genome for the species I study, but there is a sequenced genome for a related species... Can I use that?
    3. How much coverage do I need? How do I figure this out? Help!!
    4. For Plasmid Verification, do I have to provide a reference sequence or can you do de novo assembly?
    5. For mtDNA-Seq, do you have to start with isolated mtDNA? Can I just submit purified gDNA?
    6. How many samples should I submit for WGS?
    7. If my sample has RNA contamination, can you do anything to remove it?

 

1. How many cells should I use for DNA Isolation for whole genome sequencing?

For most projects, we start with ~1 million cells. This ensures we can extract enough DNA to perform full QC, library preparation, and have enough material left over in case any step needs to be repeated.

 

2. There is not a reference genome for the species I study, but there is a sequenced genome for a related species... Can I use that?

Even for very closely related species, there can be many differences between genomes. A related species can possibly be used as a guideline for assembly, but it cannot be used as a reference. 

An analogy that may better help you to understand this is a jigsaw puzzle. Think of two different puzzles, where both depict similar, but different castles. For one puzzle, you know what the completed image should look like, but for the other, you do not. If you use the reference puzzle as a guideline, you may be able to approximate the puzzle where you do not know what the completed image looks like, but you don't know how accurate this may be.  


 

3. How much coverage do I need? How do I figure this out? Help!!

This depends on your project. For de novo sequencing, we advise at least 100X coverage. For resequencing projects, some researchers only want 10-30X coverage. A member of our NGS team can help you for more detailed guidelines.


 

4. For Plasmid Verification, do I have to provide a reference sequence or can you do de novo assembly?

We can do de novo assembly without a reference sequence, although if there are duplicated or repetitive elements, a reference sequence definitely helps with the assembly process and makes it more likely that we can generate a complete assembly.


 

5. For mtDNA-Seq, do you have to start with isolated mtDNA? Can I just submit purified gDNA?

We can begin with either cells/tissues or isolated mtDNA, free of genomic DNA contamination. Because the mitochondrial genome is small, if a mixed sample is submitted and sequenced, most of the sequencing reads will belong to the gDNA, not the mtDNA. If only gDNA is provided, we cannot separate mtDNA from a mixed sample reliably, so we prefer to receive either cells as the starting material, or isolated mtDNA.


 

6. How many samples should I submit for WGS?

This depends on your project, and whether it is de novo sequencing or resequencing. For de novo, we would suggest at least two samples.


7. If my sample has RNA contamination, can you do anything to remove it?

We can do an RNase treatment, although this result in a decrease to the amount of starting material and may fragment the DNA to some extent. For samples that our team receives, if there is RNA contamination, we will discuss this with you before proceeding.

 

Got more questions? Feel free to contact our Technical Support Team at ngs@abmgood.com or leave your question in the section below. We'll be happy to help.

Topics: Next Generation Sequencing, NGS, Whole Genome Sequencing, WGS

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