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. The standard methods for mitochondrial DNA extraction have a number of limitations making them inferior solutions for NGS library preparation. Bioo Scientific has commercialized a kit which overcomes these limitations of mtDNA isolation by selectively digesting linear nuclear DNA (nDNA) while leaving circular mtDNA intact. This technology has been incorporated into the NEXTflex mtDNA-Seq Kit which includes optimized reagents for the isolation of mtDNA and for the construction of Illumina mtDNA libraries. Libraries constructed using the NEXTflex mtDNA-Seq Kit are ideal for many NGS applications including heteroplasmy analysis.

1. Simplify and Reduce Cost of
mtDNA Isolation & Library Prep

2. Mutations in mitochondrial DNA
(mtDNA) have been implicated in
various human disorders and in aging

3. Making NGS analysis of mtDNA a
priority for a number of labs

4. However, accurately determining the
diversity of mtDNA has been difficult
for a number of reasons:

5. There is only a relatively small amount of
mtDNA in each cell (<1% of the total DNA)

6. There is intercellular variability
of mtDNA content

7. mtDNA pseudogenes (Numts)
exist in nuclear DNA (nDNA)

8. Standard methods for mitochondrial
DNA (mtDNA) extraction...

9. Do not provide levels of enrichment
for mtDNA sufficient for NGS

10. Must be followed by long-range-PCR
amplification, which can bias the
sequencing results

11. Are not easily automatable

12. So what is the solution?

13. Bioo Scientific developed a library prep
kit which overcomes these limitations

14. By selectively digesting linear nuclear
DNA (nDNA) while leaving circular
mtDNA intact
Digest linear nDNA
Isolate mtDNA with
AMPure™
Beads
Repeat digestion &
cleanup steps to
ensure complete
removal of nDNA
Begin mtDNA
Library Prep
nDNA
mtDNA

15. 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 NEXTflex mtDNA-Seq Kit. Triplicate libraries were made from mtDNA isolated using the NEXTflex
mtDNA-Seq Kit (blue) or untreated gDNA (purple). mtDNA was isolated from 4 µg blood or A549 gDNA. The bars represent mean and standard deviation.

16. This technology has been incorporated
into the NEXTflex™ mtDNA-Seq Kit

17. The NEXTflex mtDNA-Seq Kit
includes optimized reagents for...

18. The isolation of mtDNA & the
construction of Illumina® compatible
mtDNA libraries for target capture or
whole mitochondrial sequencing

19. The NEXTflex™ mtDNA-Seq Kit...

20. Enriches mtDNA 100 – 350x, making it
ideal for NGS analysis

21. Reduces bias as the mtDNA does not require
long-range-PCR amplification & isolation
and library prep are easily automatable

22. Libraries constructed using the NEXTflex mtDNA-
Seq Kit are ideal for heteroplasmy analysis
100
80
60
40
20
0
ReadsalignedtomtDNA(%)
Blood HEK293
Unique Reads Aligned to the mtDNA
Genome (hg38)
HumanBloodCells
Coverage Uniformity
HEK293Cells
Performance of the NEXTflex 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) The 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.
The 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. The apparent low coverage in the beginning and the end of the sequence is an artifact caused by the inability of
Bowtie to align efficiently to circular genomes.
A B

23. 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

24. Read the whitepaper: Unparalleled
Tool for Mitochondrial DNA Analysis
DOWNLOAD WHITEPAPER

25. Learn more about how to improve your
mtDNA-Seq analysis
DOWNLOAD MANUALPURCHASE KITDOWNLOAD WHITEPAPER

26. If you have any questions email us at
nextgen@biooscientific.com
or visit our website at
BiooScientific.com/mtDNA