We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universita&#776;t Go&#776;ttingen.

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The authors have nothing to disclose.

The most critical step in the establishment of an MSPP scheme is the unequivocal genetic determination of biomarker ion identities. If it is not possible to identify a biomarker undoubtedly, then it should be excluded from the scheme13.
The C. jejuni ssp. doylei scheme includes 14 different biomarker ions. These are 5 less compared to the C. jejuni ssp. jejuni MSPP scheme13.The most significant difference between the detectable C. j...

During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has advanced to be a highly valued standard method for microbial genus and species identification in clinical microbiology1,2. Species identification is based on the recording of small protein fingerprints of intact cells or cell lysates. The typical mass range for a mass spectrometer used in routine clinical microbiology is 2-20 kDa. Additionally, the resulting spectra can be used to discriminate strains at the below-species and below-subspecies level3. Early pioneering studies have identified specific biomarker ions for a particular subgroup of strains in Campylobacter jejuni4, Clostridium difficile5, Salmonella enterica ssp. enterica serovar Typhi6, Staphylococcus aureus7-9, and Escherichia coli10-12.
The combination of several variable biomarker masses corresponding to allelic isoforms offers the option for deeper subtyping. Previously, we successfully implemented a method to convert these variations in mass profiles into meaningful and reproducible phyloproteomic relations called mass spectrometry based phyloproteomics (MSPP) on a C. jejuni ssp. jejuni isolate collection13. MSPP can be used a mass spectrometric equivalent to DNA sequence based subtyping techniques like multilocus sequence typing (MLST).
Campylobacter species are the leading cause of bacterial gastroenteritis worldwide14,15. As a consequence of Campylobacteriosis post-infectious sequela, namely, Guillain Barr&#233; Syndrome, reactive arthritis and inflammatory bowel disease can arise16. The main sources of infection are contaminated livestock meat from chicken, turkey, swine, cattle, sheep and ducks, milk and surface water15,17. Therefore, regular epidemiological surveillance studies in the context of food safety are necessary. MLST is the &#34;gold standard&#34; in molecular typing for Campylobacter species18. Because the Sanger-sequencing based MLST method is labor intensive, time consuming and relatively expensive, MLST typing is restricted to relatively small isolate cohorts. Therefore, there is a need for cheaper and faster subtyping methods. This need could be met by mass spectrometric methods like MSPP.
This paper presents a detailed protocol for MSPP-typing using a collection of Campylobacter jejuni ssp. doylei isolates and comparison of its potential with MLST.

<table border="0" cellpadding="0" cellspacing="0" width="1348">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">acetonitrile</td>
			<td style="width:663px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">34967</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoflex III TOF/TOF 200 system</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">GT02554 G201</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">bacterial test standard BTS</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">BioTools 3.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>263564</td>
			<td>Software Package</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Bruker IVD Bakterial Test Standard</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">8290190</td>
			<td>5 tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8843</td>
			<td>ATCC 49349;IMVS 1141;NCTC 11951;strain 093</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9143</td>
			<td>Goossens Z90</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG7790</td>
			<td>ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9243</td>
			<td>Goossens N130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8871</td>
			<td>NCTC A603/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9255</td>
			<td>Goossens B538</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8870</td>
			<td>NCTC A613/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar base&#160;</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1.10455 .0500</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Compass for FlexSeries 1.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>251419</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defibrinated sheep blood&#160;</td>
			<td>Oxoid Deutschland GmbH, Wesel, Germany</td>
			<td>SR0051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">ethanol</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">02854 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HCCA matrix</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Kimwipes paper tissue</td>
			<td style="width:663px;">Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MALDI Biotyper 2.0</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>259935</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mast Cryobank vials</td>
			<td>Mast Diagnostica, Reinfeld, Germany</td>
			<td style="width:119px;">CRYO/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MSP 96 polished steel target</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>224989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Mini Kit&#160;</td>
			<td style="width:663px;">Qiagen, Hilden, Germany</td>
			<td style="width:119px;">51304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">recombinant human insulin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">I2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">T6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">water, molecular biology-grade</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">W4502</td>
			<td></td>
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subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Previously, we successfully established a MSPP scheme for C. jejuni ssp. jejuni13. Here, we aimed to extend the method to the sibling subspecies C. jejuni ssp. doylei. In this specific setting, seven C. jejuni ssp. doylei isolates were acquired from the Belgian collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium. All seven isolates used for our analyses were of human origin. The genome-seque...

Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

subtyping-campylobacter-jejuni-ssp-doylei-isolates-using-mass

Research

JoVE Journal

Genetics

8.5K Views.  Universitätsmedizin Göttingen. Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

Video: Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...