Name: a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing
Uploaded: 2021-04-15T14:00:00.000+00:00
Duration: 5 min 34 s
Description: Watch this Scientific Journal Video about A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing at JoVE.com

We acknowledge funding support from the Pacific Southwest Regional Center of Excellence for Vector-Borne Diseases funded by the U.S. Centers for Disease Control and Prevention (Cooperative Agreement 1U01CK000516), CDC grant NU50CK000420-04-04, the USDA National Institute of Food and Agriculture (Hatch project 1025565), UF/IFAS Florida Medical Entomology Laboratory fellowship to Tse-Yu Chen, NSF CAMTech IUCRC Phase II grant (AWD05009_MOD0030), and Florida Department of Health (Contract CODQJ). The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service.

1. General sample storage and preparations prior to DNA extraction
<ol>
	<li>Hydrate the sample in 100 &#181;L PCR-grade water for 1 h (or overnight) at 4 &#176;C if the sample has been stored in &#62;70% alcohol to soften the tissue.</li>
</ol>
2. Sample disruption
<ol>
	<li>Set an incubator or shaking heat block at 56 &#176;C.</li>
	<li>Make proteinase K (PK) buffer/enzyme mix. 2 &#181;L of Proteinase K (100 mg/mL) and 98 &#181;L of Proteinase K Buffer (total 100 &#181;L) is required for each individual mosquito extraction. To prepare a master mix for multiple specimens, increase the total amount (combined for all individual specimens) by ~15% to ensure the availability of adequate volume.</li>
	<li>If samples were hydrated prior to extraction, discard water from each sample tube.</li>
	<li>In a 1.5 mL microcentrifuge tube containing mosquito tissue, add 100 &#181;L of PK buffer/enzyme mix.</li>
	<li>Homogenize the tissue using microcentrifuge tube pestle.</li>
	<li>Centrifuge the samples for 1 min at 9,600 x g at room temperature.</li>
	<li>Incubate the sample for 2-3 h at 56 &#176;C.</li>
	<li>During incubation, prepare the other reagents using the DNA extraction step (step 3).</li>
</ol>
3. DNA extraction
<ol>
	<li>Make a magnetic bead master mix. For each sample, mix 100 &#181;L of lysis buffer, 100 &#181;L of Isopropanol, and 15 &#181;L of magnetic beads (total 215 &#181;L). To prepare a master mix for multiple reactions, increase the total amount (combined for all individual specimens) by ~20% to ensure the availability of adequate volume.</li>
	<li>After the incubation time (step 2.7), transfer each lysate to a clean microcentrifuge tube or a microplate well using pipette.</li>
	<li>Add 100 &#181;L lysate and 215 &#181;L of the magnetic bead master mix to each sample.</li>
	<li>Use a pipette to mix it well for 10-20 s and then let it stand for 10 min at room temperature. Gently shake the tube occasionally to maximize the binding of magnetic beads and DNA.</li>
	<li>Place the tube/plate on the magnetic bead separator and wait until the solution is clear.</li>
	<li>Discard the liquid from the tube/plate using pipette. When removing the supernatant, try not to touch or disturb the magnetic beads holding the DNA.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 325 &#181;L of washing buffer 1 to each well.</li>
	<li>Mix thoroughly by pipetting and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 1 to each well.</li>
	<li>Mix thoroughly by pipetting and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 2 to each sample.</li>
	<li>Mix thoroughly and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 2 to each well.</li>
	<li>Mix thoroughly and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from the magnetic bead separator and add 100 &#181;L of elution buffer to each well.</li>
	<li>Mix thoroughly and incubate for 2 min at room temperature.</li>
	<li>Move the tube/plate over on the magnetic bead separator and wait until the solution is clear.</li>
	<li>Pipette the supernatant into a new clean 0.5 microcentrifuge tube or microplate well.</li>
	<li>Store DNA samples at 4 &#176;C (up to 1 week) or -80 &#176;C.&#160;If a microplate is being used, cover it with parafilm.</li>
	<li>Determine the DNA yields using any appropriate method. 
	NOTE: In this study, DNA fluorometer reading and absorbance at 260/280 nm were utilized.</li>
</ol>

<ol>
	<li>Nieman, C. C., Yamasaki, Y., Collier, T. C., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+extraction+protocol+for+improved+DNA+yield+from+individual+mosquitoes.">A DNA extraction protocol for improved DNA yield from individual mosquitoes.</a> F1000Research. 4, 1314 (2015).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+divergence+among+invasive+populations+of+Aedes+aegypti+in+California.">Genome-wide divergence among invasive populations of Aedes aegypti in California.</a> BMC Genomics. 20 (1), 204 (2019).</li><li>Schmidt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+conserved+CRISPR-Cas9+target+sites+within+the+highly+polymorphic+genomes+of+Anopheles+and+Aedes+mosquitoes.">Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.</a> Nature Communications. 11 (1), 1425 (2020).</li><li>Schmidt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcontinental+dispersal+of+Anopheles+gambiae+occurred+from+West+African+origin+via+serial+founder+events.">Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.</a> Communications Biology. 2, 473 (2019).</li><li>Norris, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+introgression+in+an+African+malaria+mosquito+coincident+with+the+increased+usage+of+insecticide-treated+bed+nets.">Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 815-820 (2015).</li><li>Main, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+host+preference+and+resting+behavior+in+the+major+african+malaria+vector,+Anopheles+arabiensis.">The genetic basis of host preference and resting behavior in the major african malaria vector, Anopheles arabiensis.</a> Plos Genetics. 12 (9), 1006303 (2016).</li><li>Yamasaki, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+tools+for+genomic+DNA+library+construction+of+small+insects.">Improved tools for genomic DNA library construction of small insects.</a> F1000Research. 5, 211 (2016).</li><li>Tabuloc, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+of+Tuta+absoluta+genome+to+develop+SNP+genotyping+assays+for+species+identification.">Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification.</a> Journal of Pest Science. 92, 1397-1407 (2019).</li><li>Campos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+mitogenome+sequence+of+Anopheles+coustani+from+S&#227;o+Tom&#233;+island.">Complete mitogenome sequence of Anopheles coustani from S&#227;o Tom&#233; island.</a> Mitochondrial DNA. Part B, Resources. 5 (3), 3376-3378 (2020).</li><li>Cornel, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+mitogenome+sequences+of+Aedes+(Howardina)+busckii+and+Aedes+(Ochlerotatus)+taeniorhynchus+from+the+Caribbean+Island+of+Saba.">Complete mitogenome sequences of Aedes (Howardina) busckii and Aedes (Ochlerotatus) taeniorhynchus from the Caribbean Island of Saba.</a> Mitochondrial DNA. Part B, Resources. 5 (2), 1163-1164 (2020).</li><li>Lucena-Aguilar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+source+selection+for+downstream+applications+based+on+dna+quality+indicators+analysis.">DNA source selection for downstream applications based on dna quality indicators analysis.</a> Biopreservation and Biobanking. 14 (4), 264-270 (2016).</li></ol>

The authors have nothing to disclose.

The protocol described here can be adapted for other insect species. The original version of the protocol introduced in Nieman et al.1 has been tested on multiple species, including Aedes aegypti, Ae. busckii, Ae. taeniorhynchus, Anopheles arabiensis, An. coluzzii, An. coustani, An. darlingi, An. funestus, An. gambiae, An. quadriannulatus, An. rufipes, Culex pipiens, Cx. quinquefasciatus, Cx. theileri, Drosophila suzukii, Chrysomela aeneicollis Tuta absoluta, and Keiferia lycopersicella2,8,9,10. It is expected that this protocol will work for these as well as other arthropods.
Ideally, samples should be stored in 70%-80% ethanol at 4 &#176;C prior to DNA extraction. Typically, 80% ethanol is used for storing mosquito samples in Africa. High temperature conditions could accelerate evaporation of ethanol and make ethanol content lower than what is intended (70%). Samples stored in 100% ethanol, denatured alcohol, 75%-90% isopropanol or rubbing alcohol have also resulted in successful whole genome sequencing using&#160;the Nieman et al.1 protocol, and it is expected that this protocol would perform equally well. The DNA extraction protocol described here has also been tried on samples stored dry in silica and frozen at -20 &#176;C. However, there was a higher (15%-30%) chance of failure when stored for &#62;6-12 months, suggesting that these were less than ideal storage conditions.
As DNA extraction without physical sample disruption has a higher chance of failure (33%)1, this protocol incorporates a manual physical disruption technique (step 2.5). Sample disruption can also be achieved using steel beads on a tissue lysis instrument1.
The reagents and consumable cost for DNA extraction using this protocol is comparable with other extraction methods available (Table 1). However, the protocol described here does not require an automated DNA extraction instrument. Due to manual labor involved in processing DNA extraction, one person can typically process 12-16 samples and finish with DNA quantification in a day. This is significantly less than what automated DNA extraction setup can process per day. Thus, when choosing a protocol, the sample processing capacity should be taken into account. However, the lower cost threshold this protocol offers should enable many resource limited labs and studies to leverage in high-throughput sequencing technology.
In both the Nieman et al.1 and this protocol, elution buffer can be replaced with the typical TE buffer, 10 mM Tris-Cl, or water depending on the downstream analysis. An elution buffer containing some EDTA may impact enzyme efficiency. Typical enzymatic shearing-based gDNA library preparation protocols include conditioning solutions or enhancers added to compensate for enzyme inhibition by EDTA. It should also be considered that EDTA may change the absorbance at 230 nm wavelength11. For example, higher concentrations of EDTA would create a higher absorbance value between 220-240 nm wavelength than that shown in Figure 1 (data not shown).
This protocol can be easily adapted to laboratories with minimal molecular biology tools. The protocol described here attempts to reduce the cost thresholds associated with genome extraction and thereby increase the application of high throughput sequencing to resource limited labs and studies.

Recent development of an improved DNA extraction protocol1 has allowed many high-impact downstream studies involving whole genome sequencing2,3,4,5,6. This magnetic bead-based DNA extraction protocol provides reliable DNA yield from individual mosquito samples, which in turn reduces the cost and time associated with acquiring a sufficient number of samples from field collections.
Recent advances in population and landscape genomics are directly correlated with decreasing costs of whole genome sequencing. Though the previous DNA extraction protocol1 increases efficiencies associated with high throughput sequencing, smaller labs/studies without the funds may opt out of using these new powerful landscape and population genomics tools due to costs of implementing the protocol (e.g., costs of specialized instruments).
Here, a modified DNA extraction protocol is presented that uses a similar magnetic bead extraction step as Neiman et al.1 to obtain high purity DNA but does not rely on high-cost instruments for tissue lysis and DNA extraction. This protocol is suitable for experiments requiring &#62;10 ng of high-quality DNA.

<table><tbody><tr><td>AE Buffer</td><td>Qiagen</td><td>19077</td><td>Elution buffer</td></tr><tr><td>AL Buffer</td><td>Qiagen</td><td>19075</td><td>Lysis buffer</td></tr><tr><td>AW1 Buffer</td><td>Qiagen</td><td>19081</td><td>Washing buffer 1</td></tr><tr><td>AW2 Buffer</td><td>Qiagen</td><td>19072</td><td>Washing buffer 2</td></tr><tr><td>MagAttract Suspension G</td><td>Qiagen</td><td>1026901</td><td>magnetic bead</td></tr><tr><td>Magnetic bead separator</td><td>Epigentek</td><td>Q10002-1</td><td></td></tr><tr><td>Nanodrop</td><td>ThermoFisher</td><td>ND-2000</td><td>microvolume spectrophotometer</td></tr><tr><td>PK Buffer</td><td>ThermoFisher</td><td>4489111</td><td>Proteinase K buffer</td></tr><tr><td>Proteinase K</td><td>ThermoFisher</td><td>A25561</td><td></td></tr><tr><td>Qubit</td><td>Invitrogen</td><td>Q33238</td><td>fluorometer</td></tr></tbody></table>

a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

The average DNA yield per individual mosquito head/thorax tissue was 4.121 ng/&#181;L (N = 92, standard deviation 3.513) measured using a fluorometer when eluted using 100 &#181;L of elution buffer. This is sufficient for the 10-30 ng genomic DNA input requirements necessary for whole genome library construction1,7. The quantity of DNA can vary between 0.3-29.7 ng/&#181;L depending on the mosquito body size and preservation conditions. Some of the high variability is due to the characteristic of magnetic beads used in the study. Different brands of magnetic beads may produce more consistent concentrations, as indicated in the previous report1. It should also be noted that different mosquito species differ in size and that the DNA yield is dependent on those individual and species size ranges. If the DNA concentration is below the typical ranges, one may adjust the incubation period in the proteinase K reaction (step 2.7), but this extension should be no longer than 16 h. Moreover, switching the proteinase K and/or lysis buffer brands can have significant impact on DNA yield1.
The typical microvolume fluorometer reading of the final DNA in elution buffer with 0.5 mM EDTA is shown in Figure 1. The average absorbance ratio for 260/280 nm was 2.3 (standard deviation = 0.071). The data was consistent from the samples used in the previous studies for whole genome sequencing3,4.
Typical reagent and consumable cost for extraction is around $9.50/sample. These cost estimates include reagents listed in the Table of Materials, and other consumables such as pipette tips, tubes, and gloves needed for extraction. The reagent and consumable cost are equivalent to any typical magnetic-bead-based extraction method (Table 1). The cost may vary somewhat depending on bulk purchase discounts available for any given product. The major cost benefit of this protocol comes from not requiring the automated DNA extraction instrument.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62354/62354fig01.jpg" /> 
Figure 1: Fluorometer output. Typical DNA fluorometer output of mosquito DNA eluted in elution buffer with 0.5 mM EDTA. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62354/62354fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Cost analysis for different extraction methods. The table lists the costs and the number of samples processed in one working day for different extraction methods. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62354/Table 1-62354_R1.docx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing at JoVE.com

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

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Universidad San Sebastian

Research

JoVE Journal

Biology

6.2K Views.  University of Florida. Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Video: A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Genetics

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Environment

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Immunology and Infection

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

Summary

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2D and 3D Chromosome Painting in Malaria Mosquitoes

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing