Sample Preparation Recommendations for the Chromium Genome Kit

Demonstrated Protocol, Last Modified on September 21, 2016, Permalink

DNA Quality Level and Application Performance.pdf

Method plot.pdf

Purpose and Contents

The purpose of this document is to provide guidance for HMW gDNA extraction, including general recommendations, links to protocols from 10X Genomics, and links to protocols from the literature.

  1. Overview: The Importance of High Molecular Weight Genomic DNA (HMW gDNA) -Simplified DNA Quality Table -Figure: Protocols provided by 10x Genomics, and Typical DNA Quality Level -What DNA Quality Level do I need, and how do I QC my DNA?
  2. HMW gDNA Preparation -General Advice -Summary Table: HMW gDNA Extraction Methods Sample Types -Detailed description of methods -Additional information for samples types

Overview: High Molecular Weight Genomic DNA (HMW gDNA) Results in Optimal Performance of the ChromiumTM Genome Protocols

The Chromium Genome Protocols generate long-range information across the length of individual DNA molecules. Starting the process with High Molecular Weight (HMW) genomic DNA (gDNA) will typically result in better application performance, such as increased haplotype phase block length and ability to call structural variants. For a detailed comparison of DNA Quality and application performance, please refer to our Technical Note (Link TBD). An except from this Note is shown in Figure 2.

At 10x Genomics, we report DNA size (length-weighted mean) measured after the partitioning step of the Chromium Genome workflow. The upstream workflow steps include pipetting and denaturation - both affect DNA molecule size. Therefore, upstream HMW gDNA QC (e.g. pulsed-field gel) reports a larger DNA size than seen after the workflow. The length of each individual gDNA molecule is calculated by our software pipelines. The distribution of gDNA molecule lengths, are automatically reported in Loupe (reference-based pipeline) and Supernova (de novo assembly pipeline)

On the 10X Genomics Support Website, we currently provide protocols for HMW gDNA preparation from cell suspensions, whole blood, and fresh-frozen tissue. Links to these protocols are also found through this document. This site will be periodically updated as we expand our collection of protocols.

DNA Quality Level: A simplified structure for describing gDNA samples

| DNA Quality Level | DNA Size after Chromium Genome | Comments | |--------|-------|----------|----------| | 5 | >80 kb | | | 4 | 60-80 kb | We prefer to run samples with DNA Quality Level >=4 for Supernova analysis | | 3 | 40-60 kb | We prefer to run samples with DNA Quality Level >=3 for Long Ranger analysis | | 2 | 20-40 kb | Possible to increase to DNA Quality Level 3 by using a >40 kb DNA Size Selection Protocol | | 1 | <20 kb | Possible to increase to DNA Quality Level 2 by using a >20 kb Size Selection protocol|

Figure 1. DNA Quality Level: Typical results obtained using 10x Genomics' Sample Prep Protocols

Method plot for sample prep recommendations

Figure 2. DNA Quality Level: Application performance examples

Application data example N50 Phase Block size is significantly dependent on DNA Size (above), whereas SNP sensitivity does not show a significant dependence on DNA Size (below). Data were generated using NA12878 DNA processed with Chromium Genome Rev A Protocol. Additional application examples can be found HERE(LINK)

Factors to consider while preparing your samples:

  1. Quality level of DNA based on your application goals Your application goal will determine the quality level of DNA required. Please refer to the following Technical Note for demonstrations of application performance versus DNA Quality (will be published shortly. Please contact 10x Genomics Technical Support in the mean time for guidance). This Note covers a comprehensive set of application metrics, ranging from SNP sensitivity (reference-based applications) to scaffold size (de novo assembly applications). In general, de novo assembly requires a higher DNA Quality Level than reference-based analysis. Two examples from this Note are presented in Figure 2.

  2. HMW gDNA extraction protocol We have found that the method used for gDNA prepration is strongly correlated with DNA Quality after running Chromium Genome. Mild extraction protocols (no heat or harsh buffers) produce gDNA molecules that are longer, and less damaged (ssDNA nicks, abasic sites). We strongly encourage following our gDNA extraction protocols, as they are thoroughly tested in the Chromium Genome Workflow. Figure 1 demonstrates typical results from blood, cells, and fresh-frozen tissue using 10x Genomics' Demonstrated or Validated Protocols (or in the case of CTAB extraction, a protocol kindly provided by our collaborators Allen van Deynze and Kevan Stoffel at UC Davis).

  3. QC of gDNA (optional) HMW DNA QC is challenging. We provide guidance for HMW gDNA QC at support.10xgenomics.com (HMW gDNA QC). We have found that both DNA length (which can be measured on a gel) and DNA damage (such as nicking, which cannot be measured on a gel) are important for predicting the resulting DNA Quality Level after running Chromium Genome. We have observed DNA damage with protocols that use heat or harsh chemicals such as phenol, and also with archival sample that are many years old. Therefore at 10x Genomics, we don't rely on HMW DNA QC when to deciding whether or not to run a particular sample. Rather, we use the gDNA extraction method itself as the main factor in setting expectations for application performance. We run pulsed-field gels to confirm our expectations and help with setting up a DNA Size Selection.

  4. DNA Size Selection (optional) DNA Size Selection can be used to remove DNA molecules below a certain size cutoff. We provide protocol for >20 kb and >40 kb. Is it important to note that some types of damage can cause a larger molecule to break down a bit during the Chromium Genome Workflow. DNA Size Selections are very predictable and reproducible on gDNA samples with minimal damage. Variability has been observed with archival/damaged DNA samples.

Factors to consider for using existing gDNA samples:

In many cases, customers wish to process existing gDNA samples with the Chromium Genome Assay, as opposed to preparing fresh HMW gDNA from a biological sample. Every case will be different, but here is a generalized decision process:

  1. What type of application performance are you looking for? (Technical Note will be published shortly. Please contact 10x Genomics Technical Support in the mean time for guidance)
  2. What was the method used to extract the gDNA? This can help set expectations for DNA Quality (see tables below). If you have any questions, contact 10x Genomics Technical Support (support@10xgenomics.com) for guidance.
  3. If you don't know the extraction method, do you have any QC data for your existing gDNA sample?
  4. If your sample likely won't meet the required quality, is it possible to re-extract gDNA from your biological specimen? This may not be possible, but should be considered for best results.
  5. Alternately, do you have enough gDNA (quantity and quality) for a DNA Size Selection? As noted in the table above, performing a DNA Size Selection (removing gDNA molecules <20 kb or <40 kb) can significantly increase the DNA Quality Level as described above.

HMW gDNA Preparation

Best practices for obtaining high quality HMW gDNA

Do’s: • Use a wide-bore pipet tip and pipet HMW gDNA slowly to prevent DNA from shearing • Elute and store HMW gDNA in TE or a similar buffer (not water) • Collect and store biological specimens correctly to ensure optimal DNA quality yield. DNA size selection is a great option for improving data quality if DNA quality is not optimal (link) • Use physical grinding methods while extracting HMW gDNA from tissue samples, as chemical lysis buffers can shear/ damage the DNA

Don’ts: • Avoid steps that can denature, nick or damage the DNA (heat incubation, extreme pH buffers, chemical dissociation buffers for tissue samples) • Avoid vortexing to mix samples as it can shear the DNA, use brief "pulse vortexing" only if absolutely necessary

There are a few general principles to follow for high-quality HMW gDNA isolation. We provide several gDNA preparation protocols, but for alterative protocols, please consider the following advice:

Summary Table: HMW gDNA Extraction Methods and Sample Types

Methods Recommended by 10x Genomics

|Sample Type | 10x Recommended | Kit | DNA Quality Level | Why recommended by 10x? | Requirements | Source | |--------|-------|----------|----------|----------|----------| |Cells|Yes|QIAGEN MagAttract|5|Good quality, easy protocol|1e6 cells| 10x Validated Protocol for Cells| |Cells|Yes|Promega Maxwell|3-4|Automated|5e6 cells|Write-up in progress. Contact Technical Support for a pre-print protocol| |Blood|Yes|MagAttract|5|Good quality, easy protocol|200 ul whole blood|10x Demonstrated Protocol for Whole Blood | |Blood|Yes|Promega Maxwell|3|Automated|300 ul whole blood|Write-up in progress. Contact Technical Support for a pre-print protocol| |Fresh-Frozen Tissue (Mammalian)|Yes|QIAGEN MagAttract|2|Easy protocol, but quality is not optimal for all applications|25 mg tissue| 10x Demonstrated Protocol for Fresh-Frozen Tissue| |Fresh-Frozen Tissue (Mammalian)|Yes|Promega Maxwell|2|Automated protocol, but quality is not optimal for all applications|25 mg tissue|Write-up in progress. Contact Technical Support for a pre-print protocol| |Fresh-Frozen Tissue (Single insect)|Yes|QIAGEN MagAttract|2|Easy protocol|1 insect, preferably larval|see notes below| |Tissue (Plant)|Yes|CTAB Extraction|3|High quality DNA|2g leaves|UC Davis Genome Center Website| |Tissue (General)| Yes|Gel Plug|3-5|Universal protocol, but will require sample-specific optimization. This technique is used for preparing samples for optical mapping|Varies|Zhang et al, 2012. DNA must be released from gel plug prior to running Chromium Genome, click here|

General DNA extraction methods

General Method Typical DNA Quality Notes
Column 2
Mag beads 3-5 Varies by vendor. Our recommendations are above
Gel Plug 3-5
Phenol Prep 2-3
Archival samples 1-3 Mixed/unknown methods. Damage (nicking) suspected

An outline of different methods for HMW gDNA sample preparation:

Magnetic Beads

Magnetic bead-based purification is one of the most commonly used methods to isolate gDNA from biological samples. Kits are available is many different formats, and from many different vendors. The overall workflow for magnetic bead-based gDNA preparation consists of 5 basic steps:

  1. (optional) Pre-Processing – For example, homogenization of solid tissue
  2. Lysis – Lyse cells and nuclei, liberating gDNA into solution. Lysis buffers often contain ProteinaseK and RNase
  3. Binding – Bind DNA to silica-coated magnetic beads by adding a buffer containing a chatrophic salt and isopropanol
  4. Washing – Wash magnetic beads to remove cellular debris including proteins, lipids, and sugars
  5. Elution – Elute gDNA from the magnetic beads by the addition of a slightly basic, low molarity buffer (e.g. 10 mM Tris, pH 8.5)

There are several challenges in using magnetic bead-based methods for preparing HMW gDNA >100 kb. Many protocols contain vigorous mixing and high-heat incubation steps, which can shear the DNA. We optimized QIAGEN’s MagAttract HMW (PN 67563) kit to produce HMW gDNA >200 kb (DNA Quality Lever 5 after Chromium Genome) in ~70 minutes, with only 15 minutes hands-on time. We offer HMW gDNA extraction protocols for cell suspensions, whole blood, fresh-frozen tissue, and single insects (described below).For best results, we recommend using QIAGEN’s MagAttract HMW (PN 67563) kit this particular kit, along with and our optimized protocols.

For automated HMW gDNA extraction, we recommend the Promega Maxwell Instrument. We have tested cells, blood, and tissue. The resulting DNA Quality Level is lower than our QIAGEN MagAttract protocols as presented above. Contact 10x Genomics Technical Support for a pre-print version of these protocols.

CTAB/Precipitation

HMW gDNA extraction from plants is particularly difficult sample type, due to the presence of different types of molecules that are absent in animal cells, including polyphenols and polysaccharides. This wondrous diversity of molecules across different plant species results in fragrant roses, spicy serrano peppers, and starchy potatoes. It also makes sample prep very challenging! Our collaborators at UC Davis (Kevan Stoffel, Allen van Deynze) have developed protocols for the preparation of HMW gDNA >100 kb from different plant species. These published methods involve grinding plant tissue in liquid nitrogen, followed by CTAB and chloroform clean-ups. Their general method is published and available for download: Stoffel et al., 2012. A specific implementation of this method for Chili pepper (which was sequenced at 10x using Chromium Genome and assembled with Supernova software) can be downloaded from the UC Davis Genome Center Website.

Gel Plug

This is the most common method for preparation of HMW gDNA up to and above 1 Mb in size, and has been widely used since the 1980’s (link). The basic principle of DNA extraction using gel glugs:

  1. Prepare the biological sample: Requires single cell or nuclei suspensions. Most commonly, samples are ground in liquid nitrogen and filtered to remove remaining tissue, and washed extensively via centrifugation.
  2. Embed the sample in agarose: The cell/nuclei suspension is combined with molten agarose, and cast into a mold. The solidified agarose “plugs” can then be handled easily. Agarose provides several advantages for isolating and processing HMW gDNA. First, the solid matrix formed by agarose protects the fragile chromosomal DNA from shearing (In fact, the entire chromosomes remain completely intact!) Second, the pore sizes in an agarose matrix are (~200 nM), allow for rapid diffusion of proteolytic digestion enzymes into the sample to digest cellular debris. Third, since it is easy to perform buffer exchanges, the gel plugs can be stored in a preservative solution (4C without damage for many years), and then dialyzed into TE buffer/water prior to use.
  3. Lyse and digest the cells: Depending on sample types, the content of lysis buffer will vary, but particularly contain EDTA (to chelate nucleases), detergent (to disrupt cell membranes), and Proteinase K (to digest histone proteins).
  4. Wash and store the gel plugs: At this stage, most cellular components are degraded, but the chromosomal DNA remains intact. The gel plugs are thoroughly washed to remove proteins, cells, and other debris.

The general gel plug method was first published in 1984 (Schwartz and Cantor), and a very good expansion of this method for a variety of plant species was subsequently published by Hong-Bin Zhang and Rod Wing at Texas A&M University (Zhang et al., 1995). However, our preferred source for gel plug protocols is a 2012 Nature Methods paper from Meiping Zhang and Hong-Bin Zhang, and includes specific protocols for plants, insects, and other animal tissue sources (Zhang et al, 2012).

The challenges of HMW gDNA preparation using gel plugs is that the workflow is very different from traditional DNA extractions (unfamiliar reagents and techniques), and the protocol itself takes several days from start to finish.

This is the general prep method used for optical mapping (BioNano Genomics, OpGen), so we recommending checking to see if your sample type has been analyzed with these methods and if so, using those specific protocols as a starting point.

Releasing HMW gDNA from gel plugs: In order to use the HMW gDNA for downstream applications including Chromium Genome, the agarose matrix must be enzymatically degraded. Steps for digesting agarose plugs:

  1. Equilibrate the gel plug in TE buffer. equilibrate a single gel plug in TE. Place one gel plug in a 2 ml tube filled with TE and replace the buffer several times over the course of 24 hours.
  2. Melt the gel plug. Remove the liquid buffer, leaving only the gel plug behind. Melt the plug in a 72C water bath for 1-2 minutes. Take care to work quickly and do not leave the gel plug at 72C longer than absolutely necessary to melt it.
  3. Add the enzyme Add 100 ul TE buffer and 2ul of beta-agarase (New England Biolabs, PN M0392) to the tube. Mix by slowly pipetting the contents of the tube 3X with a wide-bore pipet tip.
  4. Incubate the tube at 42C for 2 hours The HMW gDNA solution can then be stored at 4C and used directly in the Chromium Genome Workflow.

HMW gDNA Preparation: Details of sample types

Cell Suspensions

At 10X Genomics, we routinely use cultured cells to test the performance of our sample prep protocols. Cultured cells are easy to grow, and isolating HMW gDNA is straightforward and easy with our protocols. Live cells Typical DNA Quality Level: 5 Frozen cell pellets (Shipped on Dry Ice): We have received frozen cell pellets from both collaborators and vendors. The viability of cells in a frozen pellet is very low (0% to 10% viability measured by Trypan Blue staining), but we are pleased to see typical DNA Quality Level 4 from these preparations. (link) is still quite good. Typical DNA Quality Level: 4 Cryopreserved vials of cells (slow-frozen in media containing DMSO using standard protocols for cryopreservation): We have extensively tested cryopreserved cells, and found that the HMW gDNA quality obtained from our QIAGEN MagAttract protocol is equivalent to live cultured cells. Typical DNA Quality Level: 5

Whole Blood

We routinely extract HMW gDNA from whole blood. Tube type: We strongly recommend collecting blood in an EDTA vacutainer (purple cap). We have tested different EDTA tube types (vendors, volumes) and different organisms as well, and this this is a robust collection mechanism. We have tested coagulated blood (extracted into a non- EDTA tube), and found that the prepared HMW gDNA was only 30 kb on a pulsed-field gel. In this case, a blood sample was redrawn into an EDTA tube, resulting in a DNA Quality Level 5 sample. Buffy coat (instead of whole blood): We have limited experience with buffy coat samples. However, inputting 200ul of buffy coat into our recommended blood extraction protocol resulted in resulting in a DNA Quality Level 5 sample. Storage conditions

Fresh (or Fresh-Frozen) Tissue

The quality of HMW gDNA extracted from tissue depends on biological sample quality (how the sample was harvested and preserved), and dissociation method. Chemical versus physical dissociation of solid tissue The QIAGEN MagAttract protocol recommended above uses a chemical tissue dissociation buffer, whereas the gel plug protocol recommended above uses physical tissue dissociation process to release cells and DNA form the samples. In our experience, chemical dissociation buffers cause significant DNA damage, partly due to the ingredients in the buffer itself and partly due to the experimental conditions for using the buffer (high heat for >12 hours). Using a tissue dissociation buffer is easier than grinding, but the DNA quality is noticeably reduced (DNA Quality Level 2). The gel plug method referenced above uses physical dissociation (rather than chemical), and can produce DNA Quality Level 5 samples.

Single Insects

A main advantage of the Chromium Genome Workflow is that it requires a very small amount of input HMW gDNA (~1 ng), thus enabling genome assembly from very small biological samples, including single insects. We have prepared DNA from single insects using a modification of our QIAGEN MagAttract protocol for cultured cells (located in the Chromium Genome User Guide). In our hands, we observed a high degree of sample-to-sample variability, so we suggest repeating this HMW gDNA prep protocol on several different replicates and choosing the sample with the highest starting DNA quality. Single Insect HMW gDNA sample preparation (QIAGEN MagAttract)

  1. Prepare a chilled work surface by placing a metal bock on ice (we use an aluminum tube rack), covering with Parafilm, and wiping the Parafilm with 70% Ethanol.
  2. Alternatively, place a sterile Petri dish on ice
  3. Quickly, place a single insect (we used snap-frozen mosquito pupae) on top of the Parafilm, and mince the insect into a fine paste using a sterile razor blade.
  4. Quickly transfer the tissue paste into 200 ul of PBS buffer and proceed with step (d) of the "gDNA Extraction" protocol on page 8 of the Chromium Genome User Guide