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Old 09-14-2017, 09:23 PM   #1
Location: Japan

Join Date: Sep 2017
Posts: 40
Post Read coverage and accurate sample prep


I used to be in a lab that did a lot of sequencing - however, all sequencing and prepping fell on few people in the lab. Therefore I have some overview of the process of sample prepping, but need help with making sure everything is properly planned for NGS work in my new lab. My boss is asking me to calculate costs, so we can plan what is most suitable for our budget.

Material: Mitochondrial DNA (may contain miniscule traces of nDNA)
DNA amount for prepping: 0.5-1.5ng (expected)
Planned prep kit: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
(as it seems to be able to handle the very low input material that I will have)

Question 1
Since mtDNA is rather small (<20kb), you generally don't need too many reads pr. sample. However, I will be doing assessment of heteroplasmy, so I will need a good read coverage, so not under something like 3000x.

Can somebody help me with how to calculate how many reads I will need?

Question 2
Choosing indexes.
I will be running the samples on either a MiSeq or HiSeq, depending on how many samples we end up with/how many samples I can put on the MiSeq (this is a cost issue).
Will *all* indexes be equally good on either the MiSeq or HiSeq?
I understand that the MiSeq uses dual indexing as a standard, why I was thinking to buy these indexes: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
However, I am very confused about choosing the proper indexes, and would like some input.

Thank you so much for any help you can provide
Meyana is offline   Reply With Quote