bioo scientific - simplify and reduce cost of mtdna isolation and library prep
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Simplify and Reduce Cost ofmtDNA Isolation & Library Prep
Mutations in mitochondrial DNA (mtDNA) have been implicated in
various human disorders and in aging
Making NGS analysis of mtDNA a priority for a number of labs
However, accurately determining the diversity of mtDNA has been difficult
for a number of reasons:
There is only a relatively small amount of mtDNA in each cell (<1% of the total DNA)
There is intercellular variabilityof mtDNA content
mtDNA pseudogenes (Numts)exist in nuclear DNA (nDNA)
Standard methods for mitochondrial DNA (mtDNA) extraction...
Do not provide levels of enrichmentfor mtDNA sufficient for NGS
Must be followed by long-range-PCR amplification, which can bias the
sequencing results
Are not easily automatable
So what is the solution?
Bioo Scientific developed a library prep kit which overcomes these limitations
By selectively digesting linear nuclear DNA (nDNA) while leaving circular
mtDNA intact
Digest linear nDNA Isolate mtDNA withAMPure™ Beads
Repeat digestion &cleanup steps toensure complete
removal of nDNA
Begin mtDNALibrary Prep
nDNA
mtDNA
Resulting in mtDNA that has been enriched 100-350x, making it ideal for NGS analysis
NEXTflex
gDNA
NEXTflex
gDNA
0 20
% of reads mapped to mtDNA
40 60 80
Blood Cells A549 Cells
0 50 100
% of reads mapped to mtDNA
Enrichment of mtDNA reads in samples prepared using the NEXT�ex mtDNA-Seq Kit. Triplicate libraries were made from mtDNA isolated using the NEXT�ex mtDNA-Seq Kit (blue) or untreated gDNA (purple). mtDNA was isolated from 4 µg blood or A549 gDNA. �e bars represent mean and standard deviation.
This technology has been incorporated into the NEXTflex™ mtDNA-Seq Kit
The NEXTflex mtDNA-Seq Kitincludes optimized reagents for...
The isolation of mtDNA & the construction of Illumina® compatible mtDNA libraries for target capture or
whole mitochondrial sequencing
The NEXTflex™ mtDNA-Seq Kit...
Enriches mtDNA 100 – 350x, making it ideal for NGS analysis
Reduces bias as the mtDNA does not require long-range-PCR amplification & isolation
and library prep are easily automatable
Libraries constructed using the NEXTflex mtDNA-Seq Kit are ideal for heteroplasmy analysis
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Re
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tDN
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Blood HEK293
Unique Reads Aligned to the mtDNAGenome (hg38)
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Coverage Uniformity
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Performance of the NEXT�ex mtDNA-Seq Kit. mtDNA libraries were prepared from 5 human blood cell replicates (from one individual) and 6 HEK293 replicates in three independent experiments. (A) �e percentage of unique reads aligning to the mitochondrial genome are shown (mean and standard devia-
tion). Reads were aligned to human genome hg38 by Bowtie2 and duplicate reads were eliminated by Samtools. (B) Mitochondrial genome coverage uniformity. �e x and y axes represent position in the mtDNA genome and read depth respectively. Colored vertical lines represent variation compared to the reference
mitochondria sequence, suggesting heteroplasmy. �e apparent low coverage in the beginning and the end of the sequence is an artifact caused by the inability of Bowtie to align e�ciently to circular genomes.
A B
To see how libraries constructed using the NEXTflex mtDNA-Seq Kit compare to those
obtained using mtDNA isolated using Qiagen’s REPLI-g Mitochondrial DNA Kit
Read the whitepaper: Unparalleled Tool for Mitochondrial DNA Analysis
DOWNLOAD WHITEPAPER
Learn more about how to improve yourmtDNA-Seq analysis
DOWNLOAD MANUALPURCHASE KITDOWNLOAD WHITEPAPER
If you have any questions email us [email protected]
or visit our website atBiooScientific.com/mtDNA