Name: a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing
Uploaded: 2021-04-15T14:00:00
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 eac...

<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 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 lycopersicel...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 variabilit...

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 ...

This budget-friendly magnetic bead-based DNA extraction protocol makes high throughput sequencing accessible to resource limited labs and studies. This protocol does not require expensive equipment for high quality DNA extraction and it can be helpful in certain diagnostic or research situations where obtaining a sufficient quantity of DNA with a quality for high throughput sequencing is challenging. This protocol is easy to replicate with a one-time demonstration.It should be tried with a small number of samples first to get familiar with the workflow. To begin, hydrate the mosquito tissue samples stored in greater than 70%alcohol by adding 100 microliters of PCR grade water and incubating for one hour at four degrees Celsius to soften the tissue. After the incubation, discard the water and add 100 microliters of Proteinase K buffer enzyme mix.Then use a microcentrifuge tube pestle to homogenize the tissue. After homogenization, centrifuge the tissue lysate and incubate for two to three hours at 56 degrees Celsius. To extract DNA, pipette 100 microliters of the tissue lysate into a new clean microcentrifuge tube.Then add 215 microliters of the magnetic bead master mix. Using a pipette, mix the lysate and the magnetic beads mixture for 10 to 20 seconds, then let it stand for 10 minutes at room temperature. During the incubation, gently shake the tube occasionally to maximize the binding of the DNA to the magnetic beads.Next, place the tube on the magnetic bead separator until the solution becomes clear. Then using a pipette, gently aspirate the liquid from the tube without disturbing the magnetic beads holding the DNA. Move the tube away from the magnetic bead separator and wash the beads by adding 325 microliters of washing buffer one.Mix thoroughly by pipetting and incubate for one minute at room temperature. After the incubation, place the tube on the magnetic bead separator and remove the supernatant as previously demonstrated, then move the tube away from the magnetic bead separator and wash the beads once with washing buffer one. After the second wash with buffer one, wash the beads twice with 250 microliters of washing buffer two in the same manner.After the last wash, move the tube away from the magnetic bead separator and add 100 microliters of elution buffer. Mix thoroughly by pipetting and then incubate the tube for two minutes at room temperature before placing it back on the magnetic separator. When the solution becomes clear, transfer the supernatant into a new, clean 0.5 milliliter microcentrifuge tube and store appropriately.Shown here as a typical microvolume fluorometer reading of the mosquito DNA in an elution buffer containing 0.5 millimolar EDTA. The average 260 to 280 nanometer absorbance ratio is 2.3. This table shows the costs and the number of samples processed in one working day for different extraction methods.The typical reagent and consumable cost for an extraction by this protocol is around 9.50 per sample. This cost is equivalent to any typical magnetic bead-based extraction method. The major cost benefit of this protocol comes from not requiring the automated DNA extraction instrument.Make sure to change pipette tips between samples to prevent cross-contamination of DNA when working with multiple samples. We use the high quality DNA extracted using this method for whole genome sequencing. We are currently using the sequencing data to identify novel mutations in insecticide resistance or immune genes of concern in public health and disease control.Having affordable solutions for high throughput sequencing opens exciting doors for population genomics and landscape genomics aimed at understanding mosquito dispersal and gene flow which influences the success of many novel genetic control strategies.

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Research

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Biology

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

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 ...

Creating Robust Bisulfite-mRNA Libraries for Transcriptome-Wide Mapping

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

Author Spotlight: Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study 

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...