myNGS Guides
myNGS Guides MitoDeplete Kit
CRISPR-powered targeted depletion of unwanted mitochondrial DNA from next-generation sequencing libraries

Sequence more of what matters by efficiently removing overly abundant mitochondrial DNA (mtDNA) sequencing molecules from NGS libraries! The myNGSTM Guides MitoDepleteTM Kit removes of up to 95% of mtDNA-derived templates from human and mouse based NGS library pools. This simple step prior to loading the sequencer delivers 30-70% savings for ATAC-Seq sequencing, depending on the level of mtDNA contamination.

A single reaction powers mtDNA depletion of an entire sequencing lane – one pool, one reaction!

  • Process multiple libraries – One reaction is sufficient to treat an entire sequencing lane
  • Reduce sequencing costs – Significant savings for ATAC-Seq library sequencing
  • Species-specific panels – Human, Mouse, and Custom Species versions available
  • Simple protocol – Fast treatment right before sequencing
  • Any NGS library – Re-sequence old libaries at dramatically reduced cost
Ordering Information
Cat#
Qty
Price
myNGS Guides MitoDeplete -Human
myNGS Guides mtDNA Depletion Kit for Human Samples
901008
$799.00
myNGS Guides MitoDeplete -Mouse
myNGS Guides mtDNA Depletion Kit for Mouse samples
902008
$799.00
myNGS Guides MitoDeplete -Custom
myNGS Guides mtDNA Depletion kit for Custom species
909008
$1199.00
Human and Mouse available in-stock for immediate shipment, or commission a Custom kit.


Overview

CRISPR-Powered Technology

mtDNA Depletion Workflow

Fig 1. Depletion via cutting unwanted molecules. Site-specific RNPs hybridize with and then cleave molecules that are already converted to a sequencing library. Only the remaining non-cleaved molecules are sequenceable by downstream platforms, thus enriching libraries for molecules of interest.

 

Human & Mouse mtDNA

The team at Arbor Biosciences has developed panels of guide RNA’s which are highly specific to the human and mouse mitochondrial DNA with minimal off-target reactivity to the organismal genome. In human and mouse generated NGS libraries, depletion of greater than 95% mtDNA-derived templates is achievable. These catalog panels are perfect for pairing with ATAC-Seq libraries to reduce the wasted expense of sequencing contaminating mtDNA molecules. The one-step, single-tube incubation prior to sequencing is compatible with individual or pooled libraries. Easily apply to newly generated libraries or rescue previously sequenced libraries which were over-run with mtDNA to discover more valuable low-abundant sequences.

Custom Species mtDNA

Thanks to our ultra-efficient parallelized nucleic acid synthesis technology, Arbor is uniquely equipped to provide robust, affordable, expert-designed gRNA libraries of up to thousands of unique sequences, specifically for CRISPR-powered targeted mtDNA depletion in your species of choice. As with all of our genomics and synthetic biology products, we offer complimentary expert-design consultation to aid in achieving your project goals. Contact us for a custom MitoDeplete kit powered by Arbor’s myNGS Guides.

Performance

Typical Results

The myNGS Guides MitoDeplete kit is a straightforward solution for removing high amounts of mtDNA reads from NGS libraries. The below figures demonstrate typical results of both the available catalog Human and Mouse kits. If another species is of interest, contact us about developing a Custom kit for your organism.

Figure 1. Bioanalyzer® trace using Agilent dsDNA high sensitivity chip of sequencing library both before (in Red) and after (in Blue) depletion. Input 148ng of library with 33.8% Mito; incubated 1h at 37°C, column purified.

Figure 2. Normalized read coverage of human (A) and mouse (B) libraries on their mitochondrial genomes before and after depletion. Depletion reactions were performed on 100 ng human sequencing library containing 56% starting mtDNA, and 140 ng mouse sequencing library containing 36% mtDNA. After treatment, mitochondrial reads were reduced by 92% and 94%, respectively.
Resources

Protocol

Current Version: v0.8
myNGS Guides MitoDeplete Manual v0.8
FAQs

Frequently Asked Questions – myNGS Guides MitoDeplete

Can I pre-build and then store my RNP Mix prior to performing depletion?

No, we recommend using RNP Mix immediately after the 10 minute room temperature incubation.
With what kinds of libraries is the MitoDeplete kit compatible?

The kit is directly compatible with Illumina​® and Ion Torrent​® NGS libraries. However, when treating Oxford Nanopore​® or PacBio​® ​ libraries, short fragments should be removed after depletion to prevent residual fragments from occupying space on the platform flow cell.
Can I use MitoDeplete with more/fewer than 100 ng sequencing library?

Each MitoDeplete reaction can accommodate as few as 30 ng and as many as 150 ng input library under standard conditions. However the rate of nonspecific digestion depends on the total amount of mtDNA in the library, therefore optimization of the digestion time may be required for best results.
Can I use SPRI beads to clean up the depletion reaction?

Yes. You can clean up the “Depletion Reaction” by using SPRI beads according to the manufacturer’s instructions.
How can I improve the rate of mtDNA depletion?

Depletion rates are influenced by the input library amount and length distribution, the relative fraction of mitochondrial templates, the amount of Guides and Cas9, and the time and temperature of the digestion.
– For low starting mitochondrial DNA (<25%), consider reducing the incubation time from 1 hour to 30 minutes. - For high starting mitochondrial DNA (>80%), considering increasing the incubation time from 1 hour to 2 hours.
– For long-insert libraries (>500 bp average length), consider reducing the Guides to 0.5 μL per reaction and Cas9 Enzyme to 0.25 μL per reaction. Pre-dilution of both components may be required for accurate pipetting.
What are the predicted off-target effects?

The sgRNA pools included with MitoDeplete have been assayed ​in silico ​for specificity to the mitochondrial genome. Predicted off-target site lists for both human and mouse kits are available upon request to info@arborbiosci.com​.
The off-target site list overlaps with my region(s) of interest. What should I do?

We can design and synthesize custom myNGS Guides panels that avoid your target region(s) of interest while still depleting your desired non-target molecules. Please contact info@arborbiosci.com​ for more information.
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