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Old 08-29-2013, 01:15 AM   #1
Munch
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Default mtDNA sequencing using MiSeq

Hi,

I am currently planning to sequence hundreds of human mtDNA using the MiSeq platform. I have tried to use DesignStudio and SureDesign to design primers, however they both fail and i cant find any commercial kits.

I guess there is huge problems with pseudogenes and that is why nobody have developed a kit?

Is there a way to do this on a MiSeq?

Any input is very much appreciated. Thanks.

Cheers,
Munch
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Old 08-29-2013, 03:55 AM   #2
JackieBadger
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I doubt that there are no kits...

very quick google search: http://tools.invitrogen.com/content/...cms_053271.pdf
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Old 08-29-2013, 04:36 AM   #3
Munch
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Thanks for reply, but the mitoSEQr is for Sanger sequencing I am already using Sanger sequencing for mtDNA, but it is very time consuming and expensive.

What i need is a protocol or a kit for sequencing using MiSeq.
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Old 08-29-2013, 07:38 AM   #4
microgirl123
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Have you tried contacting an Illumina rep? If they don't have a kit, they can often refer you to a field scientist who can help you develop a method.
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Old 09-02-2013, 05:17 PM   #5
ScottC
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The standard Illumina Nextera/XT and TruSeq kits will work with mitochondrial DNA... or are you looking for a kit isolate the mtDNA before library preparation?
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Old 09-04-2013, 06:36 PM   #6
Genohub
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Default mtDNA

Anyone have good experience with mtDNA isolation kits or is the tried and true ultracentrifugation method the best?

Here are the kits I've heard about:

http://www.biovision.com/mitochondri...-kit-2835.html
http://www.abcam.com/mitochondrial-d...t-ab65321.html

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Old 09-19-2013, 07:29 PM   #7
rnaeye
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Hi,
NEB's microbial enrichment kit also enriches for organeller DNA enrichment since methylation density of mito DNA is very low, if exists. You may want to give a try.
https://www.neb.com/products/e2612-n...enrichment-kit

I am assuming that this might be better approach than PCR strategy especially if you are looking for deletions since it won't introduce amplification bias. Qiagen also has an amplification based mito kit. Hope this helps. I do not know how the enfichment efficiency for each method, but again NEB's method will not introduce random mutations or amplification bias for shorter melecules.
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Old 09-22-2013, 02:34 PM   #8
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Default mtDNA methylation and isolation

mtDNA is methylated, but the density may be low as you suggest. Here is a paper that describes it: DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria

The enrichment kit could deplete methylated DNA, so I'd be reluctant to use it. The other two kits from Biovision and Abcam look like they are based on isolation procedures. I'm sure they're not as clean as ultra high speed centrifugation, but I can afford a little genomic DNA contamination.

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Old 09-22-2013, 05:02 PM   #9
SNPsaurus
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Munch, my academic lab is Illumina sequencing human tumor mtDNA as two overlapping amplicons. Send me an e-mail at eajohnsn at uoregon.edu to remind me to ask my student on Monday about it and I'll send you the info.
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Old 09-22-2013, 06:06 PM   #10
JackieBadger
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Quote:
Originally Posted by SNPsaurus View Post
Munch, my academic lab is Illumina sequencing human tumor mtDNA as two overlapping amplicons. Send me an e-mail at eajohnsn at uoregon.edu to remind me to ask my student on Monday about it and I'll send you the info.


I guess, more specifically, you use long range PCR to amplify the the mtDNA genome (in two fragments) and then you use Nextera tagmentation to sequence these two fragments, and re-assemble into a consensus?

Last edited by JackieBadger; 09-22-2013 at 06:35 PM.
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Old 09-22-2013, 06:39 PM   #11
SNPsaurus
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Maybe I misunderstood the original question! But here's what we do, which is based on published protocols. We have two primer pairs, and as two separate PCR reactions (is that the part that sounded weird since you can't overlap PCR amplicons in a single reaction?) amplify the mt genome. One amplicon is from 700 to 8200, the other from 7600 to 1200. We then Nextera tagment and sequence.

We do have pretty uneven coverage (ranging from 130k reads to 12k reads, depending on the library):
Code:
human_mito	pos 8136	
		T	130321	118342	26707	22080	15382	15877	24584	12102	
		A									
		C									
		G
edit: looks like you answered your original query. Right, although just align rather than assemble. So no haplotype. But I think this was what Munch was trying to do as well.
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Last edited by SNPsaurus; 09-22-2013 at 06:42 PM.
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Old 09-23-2013, 03:31 AM   #12
JackieBadger
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Yup we have just developed primers for ~30 species to do this also. We can pool one individual from say around 10 distantly related species in to one indexing pool, and sequence maybe around ten pools. So in one run we should get 100 full length mtDNA genomes.
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Old 09-23-2013, 07:33 AM   #13
epistatic
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Qiagen's GeneRead can be used to amplify either all exons or the whole mitochondria in smaller amplicons. The two large amplicons yield more errors from the long PCR and can confound heteroplasmy measurements.

http://www.sabiosciences.com/NGS-Mitochondria.php
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Old 10-16-2013, 02:07 PM   #14
ScottC
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Illumina has a protocol too:

Quote:
This protocol explains how to prepare, sequence, and analyze the entire human
mitochondrial DNA (mtDNA) genome from clean, intact DNA samples. During sample
preparation, the mtDNA genome is amplified in two PCRs to generate two long fragments
spanning the entire human mitochondrial genome (16,569 bp). The amplicons are quantified
and pooled before library preparation. Subsequent library sequencing on the MiSeq is
followed by data analysis with MiSeq Reporter and variant calling with the mtDNA
Analysis Tool.
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