Single cell sequencing

Only a small fraction (estimated to be less than 1 %) of microbial species on Earth can be cultivated in the laboratory; thus, the standard microbial research methods based on pure culture isolation and observation can provide only very limited information about an environmental microbial community. The development and successful application of microbial small sub-unit ribosomalRNA (16S rRNA) gene PCR analysis has greatly expanded our knowledge of the diversity and phylogeny of microorganisms. Novel, yet-uncultivated microorganisms have been continually discovered by the 16S rRNA gene approach, revealing an “uncultured microbial majority”,which is estimated to comprise 40–50 as yet-uncultivated candidate phyla of bacteria and a similar number of as-yet uncultivated major lineages of archaea. Recent achievements in metagenomics (genomic sequences from the entire environmental community) and single-cell genomics are now opening the window to observation and analysis of this “biological dark matter”. Single-cell sequencing analyzes the genomic information of individual cells with the aid of rapidly advancing sequencing methodology. This technology provides the genomic information of an individual cell within its microenvironment. Generally, single-cell sequencing is comprised of two parts: single cell isolation and whole genome amplification.

Methods of Single Cell Isolation

There are three principal single-cell isolation strategies: micromanipulation, flow cytometry and microfluidics chips.

 Micromanipulation

Micromanipulation is a precise but laborious method to obtain a single cell. With the aid of micromanipulator devices, micrometer levels of precision in movement can be achieved by manually in operations such as holding, injecting and cutting cells. Glass micropipettes, optical tweezers and laser microdissection are the major tools used to manipulate single cells under the microscope.

 Cell Isolation by Glass Micropipette

In this method, a target cell is captured with a disposable glassmicropipette and then transferred to chips or tubes for subsequent analysis. The glass micropipette can be fabricated by commercial puller devices to the desired diameter. A major drawback of this method is the long distance to be covered in transferring cells from a growth or storage medium into a tube or microtiter plate (MTP) for molecular analysis. The amount of time thus required for cell transfer limits the throughput of the method. In addition, once the micromanipulator is taken out of the field of view of the microscope it is no longer possible to visually control correct transfer of the single cell or bacterium into a tube or MTP. As a result, some cells fail to transfer correctly into the bottom of a tube or MTP well.

 Cell Isolation by Optical Tweezers

Optical tweezers (OT) are capable of trapping and manipulating nanometer and micron-sized dielectric particles by exerting extremely small forces via a highly focused laser beam. In principle, a single selected cell is fixed with the laser beam and is separated from the mixed culture by moving the computer-controlled microscope stage and transferred into a predetermined separation chamber on the slide. Although this method has been successfully used in the isolation and culture of thermophilic bacteria and archaea, it has not been used for whole genome amplification and sequencing. In addition, this method may damage the target cells due to heating and photo damage, as power intensities rise to megawatts per square centimeter at the highly focused spot used for OT.

 Cell Isolation by Laser Microdissection

Laser microdissection and pressure catapulting (LMPC) is a method for isolating specific cells of interest from microscopic regions of tissue or cell samples or organisms. Cells are spread on a polyethylene membrane, and localization of the target cells, based upon their morphological or histological criteria, is performed under microscopic visualization. Then, the surrounding membrane of the target cells are cut by laser dissection. After microdissection, a laser shot of increased energy is used to catapult target cells and the surrounding membrane into a common microfuge tube positioned above the sample for genomic amplification. Cells for LMPC sorting must be suspended in deionized water and dried on a polyethylene naphthalate membrane to prevent the formation of salt crystals that can cause problems for laser microdissection and cell localization this method can isolate whole specific cells or even a single chromosomal region by cutting away the unwanted parts. In addition to genome amplification, available downstream applications include DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling.Although it offers many advantages in terms of speed, ease of use, and versatility, similar to micromanipulation, LMPC has the complication that proper cell placement into tubes or MTPs is difficult to control.

 Flow Cytometry

In flow cytometry, cells are suspended in a stream of fluid and passed by an electronic detection apparatus, allowing both analysis and sorting of up to thousands of particles per second based on multiple physical and chemical characteristics. A flow cytometer provides “high-throughput” (for a large number of cells) automated quantification of set parameters of the cell.

Flow cytometry suffers several limitations. First, the cells must be in a singlecell suspension, posing a problem in the case of microbial cells that grow in form biofilms. The parameters per cell that can be measured simultaneously is limited by the number of detectors that can be used at the same time. In practice, this number is less than two dozen. The validation of results requires the simultaneous detection of multiple markers to increase specificity. There is a surprising lack of standardization in assay and instrument set-up for flow cytometry. Standards are also lacking for how flow data are analyzed and reported. Lastly, because of the massive amount of data generated, flow cytometry data analysis can become very complicated and relies heavily on gating by a human expert.

 Microfluidic Chips

Microfluidic chips provide a useful interface for the manipulation of single cells. Cell separation and sorting on a microfluidic chip can be achieved using a variety of microscale filters and fluid dynamics mechanisms, including field-flow fractionation, hydrodynamic filtration, and inertial microfluidics. This method has been intensively applied on analysis of blood cells. The major challenge in cell sorting by microfluidic chip is to design and fabricate chips for different samples. Complications may include heterogeneous populations of cells and the presence of noncellular particles, such as sediments and minerals.

  Whole Genome Amplification Methods

Genome sequencing requires micrograms of DNA; however, single cells contain only picograms. Therefore, various methods of whole genome amplification (WGA) have been developed. Modified polymerase chain reaction (PCR) is the classic WGA method. This method requires thermocycling, random primers, degenerate or universal primers, and Taq  DNA polymerase or similar enzymes. Taq DNA polymerase lacks 30–50proofreading activity and hence has high error rates. Newer WGA methods, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) have provided improvements over PCR.

 Multiple Displacement Amplification (MDA)

Multiple displacement amplification (MDA) is a non-PCR based DNA amplification technique. MDA still uses random primers; however, this method amplifies gDNA without thermocycling and generates larger products with a lower error frequency compared with conventional PCR amplification techniques.

This method enables the rapid amplification of samples with very small amounts of DNA samples, providing a sufficient amount for genomic analysis. The reaction starts by annealing random hexamer primers to the template: DNA synthesis is carried out at a constant temperature by a high fidelity enzyme preferentially 29 DNA polymerase. This enzyme readily synthesizes DNA strands of 0.5 Mb length, and its high fidelity and 30–50 proofreading activity reduces the amplification error rate to 1 in 106–107 bases, compared to the reported error rate for conventional Taq polymerase of 1 in 9,000.

MDA generates sufficient yield of DNA products for sequencing from a single cell and is therefore a powerful tool. The large size of MDA-amplified DNA products also provides desirable sample quality for identifying the size of polymorphic repeat alleles. Its high fidelity also makes it reliable enough to be used in singlenucleotide polymorphism (SNP) allele detection. Due to the strand displacement that occurs during amplification, the amplified DNA has sufficient coverage of the source DNA, providing a high quality product for genomic analysis. The products of displaced strands can also be subsequently cloned into vectors to construct libraries for sequencing (Zhang et al. 2006). These advantages make MDA the most widely used method forWGA. The major drawback of MDA is amplification bias. Most studies on MDA have reported that this issue occurs due to over-amplification and allelic dropout. Another reported issue is that primer-primer interactions result in a sequenced product even in the absence of input template during MDA amplification. Therefore, there are problems regarding negative controls in the MDA reaction.

Multiple Annealing and Looping Based Amplification Cycles (MALBAC)

MALBAC is a PCR-based genome amplification method that introduces a step of quasilinear preamplification to reduce the bias associated with nonlinear amplification. In the preamplification phase, single-cell genomic DNA is melted at 94°C and then annealed randomly with MALBAC primers at 0°C , synthesizing semiamplicons. In the subsequent five temperature cycles, full amplicons are generated by a series quenching at 0°C, extension at 65°C, melting at 94°C and self-looping at 58°C with DNA polymerase. Self-looping of the full amplicons at the end of every cycle prevent these full amplicons from being used as a template for amplification during MALBAC, thereby reducing the amplification bias that is commonly associated with the uneven exponential amplification of DNA fragments by PCR. After the preamplification, only the full amplicons can be exponentially amplified in the following PCR using the common 27-nucleotide sequence as the primer. The PCR reaction will generate microgram levels of DNA material for sequencing experiments.

MALBAC has resulted in many significant advances over MDA amplification. MDA does not utilize DNA looping and amplifies DNA in an exponential fashion, resulting in bias. Amplification bias results in low coverage of the genome. The reduced bias associated with MALBAC has provided better genome sequence coverage, lower incidence of false positive and lower false negative mutations than other single-cell sequencing methods. However, the DNA polymerase used in the first cycle is error prone and can introduce sequencing errors that are propagated to the product DNA.

Important points before starting

This protocol is optimized for single cell material from all species of, for example, vertebrates, bacteria (gram positive and gram negative), plants (without the cell wall), sorted cells, tissue culture cells, and cells. The protocol cannot be used with fixated cells that are treated with formalin or other cross-linking agents (e.g., single cell samples obtained by laser microdissection from formalin-fixed, paraffin-embedded tissues).

Samples of 1–1000 intact cells (e.g., human or bacterial cells) are optimal for whole genome amplification reactions using the REPLI-g Single Cell Kit.

Avoid DNA contamination of reagents by using separate laboratory equipment (e.g., pipets, filter pipet tips, reaction vials, etc.). Set up the REPLI-g Single Cell reaction in a location free of DNA.

For the amplification of purified genomic DNA, refer to page 15.

REPLI-g sc DNA Polymerase should be thawed on ice. All other components can be thawed at room temperature (15–25°C).

Buffer D2 (denaturation buffer) should not be stored longer than 3 months.

DNA yields of approximately 40 μg will be present in negative (no-template) controls because DNA is generated during the REPLI-g Single Cell reaction by random extension of primer dimers, generating high-molecular-weight product. This DNA will not affect the quality of the actual samples and will not give a positive result in downstream assays.

Things to do before starting

  1. Prepare Buffer DLB by adding 500 μl H2O sc to the tube provided. Mix thoroughly and centrifuge briefly to dissolve.

Note: Reconstituted Buffer DLB can be stored for 6 months at –20°C. Buffer DLB is pH-labile.

  1. All buffers and reagents should be vortexed before use to ensure thorough mixing.
  2. Set a water bath, heating block, or a programmable thermal cycler to 30°C.
  3. If a thermal cycler is used with a heated lid, the temperature of the lid should be set to 70°C.

Procedure

  1. Prepare sufficient Buffer D2 (denaturation buffer) for the total number of whole genome amplification reactions (Table 4).

Note: The total volume of Buffer D2 given in Table 4 is sufficient for 12 reactions. If performing fewer reactions, store residual Buffer D2 at –20°C. Buffer D2 should not be stored longer than 3 months.

  1. Place 4 μl cell material (supplied with PBS) into a microcentrifuge tube. If using less than 4 μl of cell material, add PBS sc to bring the volume up to 4 μl.

Note: The amount of PBS sc supplied with the REPLI-g Single Cell Kit is insufficient to prepare serial dilutions of cell material.

Alternatively, 0.5 μl whole blood can be used.

  1. Add 3 μl Buffer D2. Mix carefully by flicking the tube and centrifuge briefly.

Note: Ensure that the cell material does not stick to the tube wall above the buffer line.

  1. Incubate for 10 min at 65°C.
  2. Add 3 μl Stop Solution. Mix carefully by flicking the tube and centrifuge briefly. Store on ice.
  3. Thaw REPLI-g sc DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly.

The REPLI-g sc Reaction Buffer may form a precipitate after thawing. The precipitate will dissolve by vortexing for 10 s.

  1. Prepare a master mix. Mix and centrifuge briefly.
  2. For each reaction, add 40 μl master mix to 10 μl denatured DNA (from step 5).
  3. Incubate at 30°C for 8 h.

After incubation at 30°C, heat the water bath or heating block up to 65°C if the same water bath or heating block will be used in step 10.

  1. Inactivate REPLI-g sc DNA Polymerase at 65°C for 3 min.
  2. If not being used directly, store amplified DNA at 4°C for short-term storage or –20°C for long-term storage.
  3. Use the correct amount of REPLI-g amplified DNA diluted in water or TE according to the manufacturer’s instructions. If performing PCR analysis, dilute an aliquot of amplified DNA 1:100 and use 2 μl of diluted DNA for each PCR reaction.
  4. Amplified DNA can be used in a variety of downstream applications, including next-generation sequencing, array CGH, and quantitative PCR.

Protocol: Amplification of Purified Genomic DNA

Important points before starting

This protocol is optimized for whole genome amplification from >10 ng of purified genomic DNA template. The template DNA should be suspended in TE. If the DNA is of sufficient quality (e.g., high-molecular-weight DNA with no inhibitors [e.g., detergents or organic solvents]), smaller amounts (1–10 ng for eukaryotic DNA or 10–100 pg for bacterial DNA) may be used.

Avoid DNA contamination of reagents by using separate laboratory equipment (e.g., pipets, filter pipet tips, reaction vials, etc.). Set up the REPLI-g Single Cell reaction in a location free of DNA.

For direct amplification of DNA from cell material, see page 12.

For best results, the template DNA should be >2 kb in length with some fragments >10 kb.

REPLI-g sc DNA Polymerase should be thawed on ice (see step 6). All other components can be thawed at room temperature (15–25°C).

Buffer D1 (denaturation buffer) and Buffer N1 (neutralization buffer) should not be stored longer than 3 months.

DNA yields of approximately 40 μg will be present in negative (no-template) controls because DNA is generated during the REPLI-g Single Cell reaction by random extension of primer dimers, generating high-molecular-weight product. This DNA will not affect the quality of the actual samples and will not give a positive result in downstream assays.

Things to do before starting

Prepare Buffer DLB by adding 500 μl H2O sc to the tube provided. Mix thoroughly and centrifuge briefly to dissolve.

Note: Reconstituted Buffer DLB can be stored for 6 months at –20°C. Buffer DLB is pH-labile.

All buffers and reagents should be vortexed before use to ensure thorough mixing.

Set a water bath or heating block to 30°C.

Procedure

  1. Prepare sufficient Buffer D1 (denaturation buffer) and Buffer N1 (neutralization buffer) for the total number of whole genome amplification reactions (Tables 6 and 7).
  2. Place 2.5 μl template DNA into a microcentrifuge tube. The amount of template DNA should be >10 ng. A DNA control reaction can be set up using 10 ng (1 μl) control genomic DNA (e.g., REPLI-g Human Control Kit, cat. no. 150090). Adjust the volume by adding PBS sc (provided) to the starting volume of your sample.
  3. Add 2.5 μl Buffer D1 to the DNA. Mix by vortexing and centrifuge briefly.
  4. Incubate at room temperature for 3 min.
  5. Add 5.0 μl Buffer N1. Mix by vortexing and centrifuge briefly. Store on ice.
  6. Thaw REPLI-g sc DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly. The REPLI-g sc Reaction Buffer may form a precipitate after thawing. The precipitate will dissolve by vortexing for 10 s.
  7. Prepare a master mix according Table 8. Mix and centrifuge briefly.
  8. Add 40 μl master mix to 10 μl denatured DNA (from step 5).
  9. Incubate at 30°C for 8 h. Maximum DNA yield is achieved using an incubation time of 16 h. After incubation at 30°C, heat the water bath or heating block up to 65°C if the same water bath or heating block will be used in step 10.
  10. Inactivate REPLI-g sc DNA Polymerase for 3 min at 65°C.
  11. If not being used directly, store amplified DNA at 4°C for short-term storage or –20°C for long-term storage. DNA amplified using the REPLI-g Single Cell Kit should be treated as genomic DNA with minimal freeze-thaw cycles. We therefore recommend storage of nucleic acids at a concentration of at least 100 ng/μl.
  12. Use the correct amount of REPLI-g amplified DNA diluted in water or TE according to the manufacturer’s instructions. If performing PCR analysis, dilute an aliquot of amplified DNA 1:100 and use 2 μl of diluted DNA for each PCR reaction.
  13. Amplified DNA can be used in a variety of downstream applications, including next-generation sequencing, array CGH, and quantitative PCR.