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Here we present a protocol to lyse cyanobacteria and green algae single cells that allows for subsequent single-cell whole genome amplification in a microfluidic platform with a 100% success rate.
Single-cell sequencing is becoming popular for analyzing the genome of a single cell within a heterogenous cell population. It often relies on microfluidic tools to perform single cell isolation and nanoscale chemical reactions to lyse the cell and amplify its genome. However, single-cell sequencing has mainly been applied to human cells and certain bacterial species that are easy to lyse. It is still rare to use single-cell sequencing in environmental studies, as many species of vital environmental significance such as cyanobacterial and green algal species have complex and rigid cell wall structures. To extend single-cell sequencing to these hard-to-lyse species, it is essential to develop an effective lysis method compatible with microfluidic tools and amplification chemistry. Here, we present a lysis method proven effective for cyanobacterial and green algal species for subsequent microfluidic-based single-cell whole genome sequencing (SC-WGA). The protocol combines thermal and chemical lysis mechanisms and has achieved >25 ng DNA for 100% of the single cells after on-chip amplification. Nostoc was chosen as a cyanobacterial model for the protocol development. The optimized protocol was directly applied to Gloeocapsa, another cyanobacterial species, and Sphaerocystis, a eukaryotic green alga, without modifications and achieved a 100% success rate.
Single-cell whole genome sequencing (SC-WGS) has been popular for studying the genetic heterogeneity of complex cell communities on a cellular level1,2,3. Generally, SC-WGS requires single-cell isolation, lysis, and amplifying the femtograms to picograms of genomic DNA to generate enough DNA for standard library preparation (>25 ng)4,5. Microfluidics is an ideal tool for SC-WGA, as it handles nanoscale of fluid precisely6,7,8,9,10,11,12, permitting single cell isolation into microchambers for lysis and genome amplification13,14. Multiple displacement amplification (MDA)15 has been a common chemistry technique in microfluidic-based SC-WGA. It uses φ29 DNA polymerase and random primers to produce copies of template DNA with relatively high fidelity and low error rates, and it is easy to implement in most microfluidic systems16,17,18.
So far, SC-WGS has been mostly performed on human cells, as they can be simply lysed and processed following most SC-WGA kit instructions. Bacterial SC-WGA is more challenging due to rigid and multi-layered cell walls19 and 1000x less the amount of starting DNA20. Even so, efforts have been directed to bacterial SC-WGA, as these microorganisms21 are being increasingly recognized as critical factors in human microbiome22,23. However, SC-WGS is still rare in species significant to environmental studies including cyanobacteria24,25 and green algae26,27 due to complex and thick cell structures that are often enveloped in extracellular matrix and thus hard to lyse.
Standard methods for mechanically disrupting rigid species such as bead-beating, sonication, and lyophilization are not compatible with microfluidic tools28. Chemical lysis is better suited for SC-WGA in microfluidics; however, common chemical lysis agents such as sodium dodecyl sulfate and sarkosyl have substantial deleterious effect on polymerase activities in MDA chemistry29. Other effective chemicals including phenol and spermine are toxic and need to be used in a fume hood; thus, they are not generally applicable in microfluidics19. Freeze-thawing followed by alkaline treatment is efficient for cyanobacteria lysis for SC-WGA in well plates30, but it requires placing the plates in a -80 °C freezer for 1 h and thus is not implementable in microfluidic systems with various control units.
A common alternative is to pre-lyse a cyanobacterial or green algal cell population in-tube using ionic surfactants followed by wash steps before injecting the cell suspension into microfluidic chips29, 31. However, this approach is not applicable for cells in low abundance and will likely release DNA from the bulk cell into the extracellular milieu, leading to contamination in the SC-WGA step. Multi-round amplification is another common option when cells cannot be sufficiently lysed, but it often leads to increased bias in the sequencing data.
To enable SC-WGA for cyanobacterial and green algal cells without the aforementioned concerns, present here is a method effective for the lysis of single cyanobacterial and green algal cells for subsequent MDA-based SC-WGA in microfluidic devices, which generates >25 ng of genomic DNA per cell. This approach is based on three major cell lysis methods including thermal30,32,33, enzymatic34,35, and chemical lysis36,37,38. In this work, the species Nostoc was used as a model because of substantial lysis difficulties encountered in earlier studies as a cyanobacterial species19,39. Its cell wall primarily consists of an external layer of exopolysaccharide and polymerized proteins, outer membrane, thick peptidoglycan layer, and inner cytoplasmic membrane40. Our lysis method was designed to systematically degrade these cells from their outermost to innermost layers without suppressing MDA chemistry. This protocol was tested on another cyanobacterial species, Gloeocapsa, and green algal species (Chlorophyta) Sphaerocystis, whose cell wall is often composed of microfibrillar polysaccharides and enveloped in polysaccharides41,42. Despite the existence of various novel high-throughput microfluidic-based single cell isolation technologies such as droplet-based fluorescent-activated cell sorting13 and in-gel single cell trapping in virtual microfludics43, laser tweezers were chosen for single cell trapping and transport due to its higher precision and single-cell confidence, which is especially important for isolating target cells in samples with complex components. 100% success was achieved for all three species following the optimized protocol without further efforts. We believe that this effective lysis method may enable SC-WGA of cyanobacterial and green algal cells in microfluidics for a wide range of single cell genomic studies in environmental research.
1. Preparation of desiccated cell species (Nostoc, Gloeocapsa, Sphaerocystis)
2. Lysis buffer preparation
3. Set-up of microfluidic device for single cell experiment
4. Cell sorting in microfluidic device using laser tweezers
5. SC-WGA in microfluidic device
6. Collecting on-chip SC-WGA products
The protocol was developed in our optofluidic platform at the Mayo Clinic21. This platform consists of a microscope, optical tweezers, and microfluidic chip that support the serial addition of reagents (Figure 1A-D). Figure 2 illustrates the cell wall structures of the cyanobacterial and green algal species tested using the protocol. Figure 3 shows the overall...
During the process of single cell isolation in a microfluidic device using laser tweezers, it is essential to ensure that no undesired cells are in the cell isolation chambers prior to adding lysis buffers. Undesired cells should be moved out of the chamber to minimize contaminating DNA caused by these cells. Earlier studies have shown that lasers with wavelength between 1250 nm and 1550 nm with 100 mW power can increase the temperature and rupture the cell membrane44. However, other evidence has ...
The authors have nothing to disclose.
This research was funded by the following sources: Marina Walther-Antonio and Yuguang Liu acknowledge The Ivan Bowen Family Foundation and CTSA Grant Number KL2 TR002379 from the National Center for Advancing Translational Science (NCATS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Dirk Schulze-Makuch and Jean-Pierre de Vera acknowledge support from ERC Advanced Grant "HOME" (#339231). In addition, Jean-Pierre de Vera acknowledges support from ESA for the BIOMEX project (ESA-ILSRA 2009-0834) and DLR for the DLR-FuW-Project BIOMEX (2474128). Charles S. Cockell was supported by the Science and Technology Facilities Council (STFC), Grant No. ST/M001261/1.
We thank the Mayo Clinic Microbiome Program and the Mayo Clinic Center for Individualized Medicine for support. We also thank Minnesota Nano Center at the University of Minnesota (Minneapolis, MN, USA) and Dr. Alexander Revzin at Mayo Clinic for granting us access to their microfabrication facilities.
Name | Company | Catalog Number | Comments |
DTT | Bio-rad | 1610611 | |
EDTA | Thermo Fisher | 15575020 | pH 8.0 |
Lysozyme | Epicentre | R1804M | |
MATLAB | microfluidic user interface | ||
Microscope | Nikon | Eclipse/Ti | |
Nuclease-free water | Thermo Fisher | AM9938 | |
Optical Tweezers | Thorlabs | OTM 211 | |
PBS | Thermo Fisher | 10010023 | pH 7.4 |
PDMS | Dow Coring | Sylgard 184 | |
Pluronic F-127 | Sigma Aldrich | 9003116 | |
Single cell WGA kit | Qiagen | 150343 | Include D2 buffer and neutralization buffer |
Tapestation | Agilent | 2200 | |
Tween 20 | Sigma Aldrich | 9005-64-5 |
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