- Enables targeted sequencing of the mitochondrial genome
- Offers efficient mtDNA isolation and library construction in a single workflow
- Produces a greater number of unique mtDNA reads compared to traditional PCR
- Enables heteroplasmy analysis
- Allows for increased multiplexing
The NEXTflex™ mtDNA-Seq Kit includes optimized reagents for the isolation of mitochondrial (mtDNA) and construction of Illumina-compatible paired-end or single read libraries from 2 – 4 µg of genomic DNA. This kit was developed using technology published in the journal, Nucleic Acids Research: Stable Heteroplasmy at the Single-cell Level is Facilitated by Intercellular Exchange of mtDNA.
mtDNA Isolation Procedure
The NEXTflex mtDNA-Seq Kit enables selective enrichment of mitochondrial DNA from total genomic DNA samples, without the need for prior mitochondrial DNA isolation using CsCl gradients. The NEXTflex mtDNA-Seq Kit selectively digests linear nuclear DNA (nDNA) while leaving circular mitochondrial DNA intact; enriching the mitochondrial genome 100-350 times.
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 (red) or untreated gDNA (purple). mtDNA was isolated from 4 µg blood or A549 gDNA. The bars represent mean and standard deviation.
Optimized Library Prep for Mitochondrial DNA Sequencing
The mtDNA-Seq Kit incorporates a library prep protocol designed for low input samples and produces Illumina-compatible single read or paired-end mtDNA-Seq libraries. Using the NEXTflex™ ChIP-Seq Barcodes, the user can multiplex 2 - 96 samples at once providing unparalleled multiplexing capacity.
Improved Mitochondrial DNA Sequencing Data
Libraries prepared using the NEXTflex mtDNA-Seq Kit produce a greater number of unique reads reducing the number of sequencing reads required per sample. More information can be found in: Unparalleled Tool for Mitochondrial DNA Analysis, a paper comparing sequencing results obtained using the NEXTflex mtDNA-Seq Kit to those obtained using the Qiagen REPLI-g Mitochondrial DNA Kit.
mtDNA libraries were prepared from 5 human blood cell replicates and 6 HEK293 replicates. (A) The percentage of unique reads aligning to the mitochondrial genome are shown (mean and standard deviation). (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 suggest heteroplasmy.
Publications that Cite Using the NEXTflex mtDNA-Seq Kit
Jayaprakash, A. D., et al. (2015) Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA. Nucl. Acids Res. doi: 10.1093/nar/gkv052.
The NEXTflex mtDNA-Seq Kit contains enough material to prepare 8 or 48 DNA samples for Illumina® compatible mitochondrial DNA sequencing. The shelf life of all reagents is 12 months when stored properly. 6X Loading Dye, Nuclease-free Water, and Resuspension Buffer can be stored at room temperature. All other components should be safely stored at -20°C. These kits ship on dry ice.
NEXTflex™ mtDNA Buffer Mix 1
NEXTflex™ mtDNA Buffer Mix 2
NEXTflex™ Nuclear DNA Digest Mix
NEXTflex™ mtDNA Primer Mix
NEXTflex™ Nuclear DNA Primer Mix
NEXTflex™ mtDNA End Repair Buffer Mix
NEXTflex™ mtDNA End Repair Enzyme Mix
NEXTflex™ mtDNA Adenylation Mix
NEXTflex™ mtDNA Ligation Mix
NEXTflex™ ChIP Adapter
NEXTflex™ Primer Mix
NEXTflex™ PCR Master Mix
6X Loading Dye
MW 100 bp Ready-to-Load Ladder
Required Materials Not Provided
2 - 8 μg of genomic DNA in up to 35 μL nuclease-free water
(Optional)NEXTflex™ ChIP-Seq Barcodes – 6 , 12 , 24 , 48 or NEXTflex-96™ ChIP-Seq Barcodes (Cat # 514120, 514121, 514122, 514123, 514124)
Ethanol 100% (room temperature)
Covaris System (S2, E210) or other device for fragmenting DNA
96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
Magnetic Stand-96 (Life Technologies, Cat # AM10027) or similar
Heat block at 37°C and at 70°C
2, 10, 20, 200 and 1000 μL pipettes, multichannel pipettes
Nuclease-free barrier pipette tips
1.5 mL nuclease-free microcentrifuge tubes
Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ºC (Boston Bioproducts, Cat # P-730)
1X TAE buffer
SYBR Gold (Invitrogen, Cat # S11494)
UV transilluminator or gel documentation instrument
Gel electrophoresis apparatus
Electrophoresis power supply