This work has received funding from the European Union&#39;s Horizon 2020 research and innovation programme under grant agreement No. 643476 (COMPARE).

NOTE: List of software tools to be used: usearch v11.0.667; muscle v3.8.1551; porechop 0.2.4; cutadapt 2.5; minimap2 2.16-r922; samtools 1.9; trimmomatic 0.39; bbmap 38.33; spades v3.13.1; kma-1.2.8
1. Primer design
<ol>
	<li>Start with downloading or retrieving a set of relevant reference whole genome sequences from public or private data collections. For instance, retrieve all full length USUV genomes (taxid64286) from the NCBI database22. USUV encodes a genome of around 11,000 nucleotides so only retrieve the sequences with a sequence length of 8,000-12,000 nucleotides. Do this using the following search entry: 
	- taxid64286[Organism:noexp] AND 8000[SLEN]:12000[SLEN].
	<ol>
		<li>Click on Send to | Complete Record | File; use Format = FASTA and create the File.</li>
	</ol>
	</li>
	<li>To downsize the set of reference sequences, remove duplicate sequences or sequences with over 99% nucleotide identity from the dataset. Do this using the cluster fast option from usearch23. On the command line enter: 
	- usearch -cluster_fast All_USUV.fasta -id 0.99 -centroids All_USUV_dedup.fasta</li>
	<li>To generate the primers, sequences need to be aligned. This is done using MUSCLE24. On the command line enter: 
	- muscle -in All_USUV_dedup.fasta -out All_USUV_dedup_aligned.fasta -log log_muscle.txt 
	NOTE: It is essential to manually inspect the alignment to check for discrepancies. These can be manually corrected if needed and the ends can be trimmed according to the length of most whole genome sequences.</li>
	<li>Primal is used to make a draft selection of the primers which can be used for full length amplicon sequencing19. Upload the alignment to the primal website (<a href="http://primal.zibraproject.org/" target="_blank">http://primal.zibraproject.org/</a>) and select the preferred amplicon length and overlap length between the different amplicons. Go to primal.zibraproject.org, fill in the Scheme name, upload the aligned fasta file, select the amplicon length, overlap size, and generate the scheme.</li>
	<li>Align the complete set of available complete USUV sequences (not the downsized or deduplicated set). On the command line enter: 
	- muscle -in All_USUV.fasta -out All_USUV_aligned.fasta -log log_muscle.txt 
	NOTE: Map the generated primers against the complete alignment (do not use the deduplicated alignment), manually correct errors and include a maximum of 5 degenerative primer positions.</li>
</ol>
2. Multiplex PCR
<ol>
	<li>Perform the multiplex PCR using the designed primers and nanopore and Illumina sequencing. The multiplex PCR for USUV was performed as previous described19,21.</li>
	<li>Perform basecalling with flip-flop version 3.0.6.6+9999d81.</li>
</ol>
3. Data analysis to generate consensus sequences from nanopore data
<ol>
	<li>Several samples can be multiplexed on a single nanopore sequencing run. After performing the sequence run, demultiplex the nanopore data. Use Porechop25 for this. To prevent contamination and enhance accuracy, use the require_two_barcodes flag. On the command line enter: 
	- porechop -i Run_USUV.fastq -o Run_USUV_demultiplex --require_two_barcodes</li>
	<li>After demultiplexing, remove primer sequences (indicated in the file Primers_Usutu.fasta in both orientations) using cutadapt26. In addition, remove sequences with a length shorter than 75 nucleotides. The primers have to be removed since they can introduce artificial biases in the consensus sequence. On the command line enter: 
	- cutadapt -b file:Primers_USUV.fasta -o BC01_trimmed.fastq BC01.fastq -m 75</li>
	<li>Demultiplexed sequence reads can be mapped against a panel of distinct reference strains using minimap227 and a consensus sequence can be generated using samtools28. Follow the example below which shows the procedure of a reference-based alignment and the consensus sequence generation of one sample: BC01. On the command line enter: 
	- minimap2 -ax map-ont Random_Refs_USUV.fasta BC01_trimmed.fastq &#62; BC01.bam 
	- samtools sort BC01.bam &#62; BC01_sorted.bam 
	- bcftools mpileup -Ou -f Random_Refs_USUV.fasta BC01_sorted.bam | bcftools call -mv -Oz -o BC01.vcf.gz 
	- bcftools index BC01.vcf.gz 
	- cat Random_Refs_USUV.fasta | bcftools consensus BC01.vcf.gz &#62; BC01_consensus.fasta</li>
	<li>For reference-based alignments it is essential that a closely related reference sequence is used. Therefore, perform a BlastN search with the generated consensus sequence to identify the closest reference strain. After that, repeat the reference-based alignment with the closest reference strain as reference (step 3.3 and 3.4). On the command line enter: 
	- minimap2 -ax map-ont Ref_USUV_BC01.fasta BC01_trimmed.fastq &#62; BC01_ref.bam 
	- samtools sort BC01_ref.bam &#62; BC01_sorted_ref.bam 
	- bcftools mpileup -Ou -f Ref_USUV_BC01.fasta BC01_sorted_ref.bam | bcftools call -mv -Oz -o BC01_ref.vcf.gz 
	- bcftools index BC01_ref.vcf.gz 
	- cat Ref_USUV_BC01.fasta | bcftools consensus BC01_ref.vcf.gz &#62; BC01_ref_consensus.fasta</li>
</ol>
4. Analysis of the Illumina data
<ol>
	<li>These sequences are automatically demultiplexed after sequencing. Reads can be quality controlled using trimmomatic29. For paired-end Illumina sequences, use the commonly used cut-off median PHRED score of 33 and a minimal read length of 75 to get accurate, high quality reads. On the command line enter: 
	- trimmomatic PE -phred33 9_S9_L001_R1_001.fastq.gz 9_S9_L001_R2_001.fastq.gz 9_1P.fastq 9_1U.fastq 9_2P.fastq 9_2U.fastq LEADING:3 TRAILING:3 SLIDINGWINDOW:3:15 MINLEN:75</li>
	<li>Remove primers (indicated in the file Primers_Usutu.fasta in both orientations), since they can introduce artificial biases, using cutadapt26. In addition, remove sequences with a length shorter than 75 nucleotides using the commands below. On the command line enter: 
	- cutadapt -b o 9_1P_trimmed.fastq -p 9_2P_trimmed.fastq 9_1P.fastq 9_2P.fastq -m 75</li>
	<li>Before de novo assembly, the sequence reads can be normalized for an even coverage across the genome. This is essential since de novo assemblers like SPAdes take the read coverage into account when assembling sequence reads. Normalize reads to a read coverage of 50 using BBNorm from the BBMap package30. On the command line enter: 
	- bbmap/bbnorm.sh target=50 in=9_1P_trimmed.fastq in2=9_2P_trimmed.fastq out=Sample9_FW_norm.fastq out2=Sample9_RE_norm.fastq</li>
	<li>The normalized reads are de novo assembled using SPAdes31. Default settings are used for the assembly using all different kmers (21, 33, 55, 77, 99 and 127). On the command line enter: 
	- spades.py -k 21,33,55,77,99,127 -o Sample9 -1 Sample9.qc.f.fq -2 Sample9.qc.r.fq</li>
	<li>Map the QC reads against the obtained consensus sequence using minimap2 and programs like Geneious, Bioedit or Ugene to curate the alignment. It is important to check the beginning and the end of the contig.
	<ol>
		<li>Align the QC reads against the obtained consensus sequencing using minimap2.</li>
		<li>Import the alignment in Geneious/Bioedit/UGene.</li>
		<li>Manually inspect, correct and curate especially the beginning and the end of the genome.</li>
	</ol>
	</li>
</ol>
5. Determining the required read coverage to compensate for the error profile in nanopore sequencing using Illumina data as gold standard
<ol>
	<li>Select sequence reads mapping to one amplicon, in this case amplicon 26. Subsequently, map the nanopore reads against this amplicon using minimap2. Use Samtools to select only the reads mapping to amplicon 26 and to convert the bam file into fastq. On the command line enter: 
	- minimap2 -ax map-ont -m 150 Amplicon26.fasta BC01_trimmed.fastq &#62; BC01.bam 
	- samtools view -b -F 4 BC01.bam &#62; BC01_mapped.bam 
	- samtools bam2fq BC01_mapped.bam | seqtk seq - -&#62; BC01_mapped.fastq</li>
	<li>Randomly select subsets of for instance 200 sequence reads one thousand times. For example, changing it to 10 will result in the random selection of one thousand times a subset of 10 sequence reads. The script is provided as Supplementary File 1. On the command line enter: 
	- python Random_selection.py</li>
	<li>All randomly selected sequence reads are aligned to amplicon 26. Use KMA32 to map the sequence reads and to immediately generate a consensus sequence. Use optimized settings for nanopore sequencing, indicated by the -bcNano flag. On the command line enter: 
	- kma index -i Amplicon26.fasta 
	- for file in random_sample*; do 
	- sampleID=${file%.fastq} 
	- kma -i ${sampleID}.fastq -o ${sampleID} -t_db Amplicon26.fasta -mem_mode -mp 5 -mrs 0.0 -bcNano 
	- done</li>
	<li>Inspect the generated consensus sequences on the command line using: 
	- cat *.fsa &#62; All_genomes.fsa 
	- minimap2 -ax map-ont Amplicon26.fasta All_genomes.fsa &#62; All_genomes.bam 
	- samtools sort All_genomes.bam &#62; All_genomes_sorted.bam 
	- samtools stats All_genomes_sorted.bam &#62; stats.txt
	<ol>
		<li>The error rate is displayed in the stats.txt under the heading error rate #mismatches / bases mapped. Display it on the screen with the following command: 
		- grep ^SN stats.txt | cut -f 2-</li>
		<li>The amount of indels is displayed under the heading #Indels per cycle. Display it on the screen with the following command: 
		- grep ^IC stats.txt | cut -f 2-</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Nanopore sequencing is constantly evolving and therefore there is a need for methods to monitor the error rate. Here, we describe a workflow to monitor the error rate of the nanopore sequencer. This can be useful after the release of a new flow cell, or if new releases of the basecalling are released. However, this can also be useful for users who want to set-up and validate their own sequencing protocol.
Different software and alignment tools can yield different results33. In this manuscript, we aimed to use freely available software packages which are commonly used, and which have clear documentation. In some cases, preference might be given to commercial tools, which generally have a more user-friendly interfaces but have to be paid for. In the future, this method can be applied to the same sample in case big modifications in sequence technology or basecalling software are introduced Preferentially this should be done after each update of the basecaller or flowcell, however given the speed of the current developments this can be also been done only after major updates.
The reduction in the error rate in sequencing allows for a higher number of samples to be multiplexed. Thereby, nanopore sequencing is getting closer to replacing conventional real time PCRs for diagnostic assays, which is already the case for influenza virus diagnostics. In addition, the reduction of the error rate increases the usability of this technique sequencing, for instance for the determination of minor variants and for high-throughput unbiased metagenomic sequencing.
A critical step in the protocol is that close, reliable reference sequences need to be available. The primers are based on the current knowledge about virus diversity and might need to be updated every once in a while. Another critical point when setting up an amplicon-based sequencing approach is the balancing of the primer concentration to get an even balance in amplicon depth. This enables the multiplexing of more samples on a sequence run and results in a significant cost reduction.

Fast developments in third generation sequence technologies allows us to move forward towards close to real-time sequencing during viral outbreaks. This timely availability of genetic information can be useful to determine the origin and evolution of viral pathogens. Gold standards in the fields of next generation sequencing however, are still the second-generation sequencers. These techniques rely on specific and time-consuming techniques like clonal amplification during an emulsion PCR or clonal bridge amplification. The third-generation sequencers are cheaper, hand-held and come with simplified library preparation methodologies. Especially the small size of the sequence device and the low purchase price makes it an interesting candidate for deployable, fieldable sequencing. This could for instance be seen during the Ebola virus outbreak in Sierra Leone and during the ongoing arbovirus outbreak investigations in Brazil1,2,3. However, the reported high error rate4 might limit the applications for which nanopore sequencing can be used.
Nanopore sequencing is evolving quickly. New products are available in the market on a regular basis. Examples of this are for instance the 1D squared kits which enables sequencing of both strands of the DNA molecule, thereby boosting the accuracy of the called bases5 and the development of the R10 flow cell which measures the change in current at two different instances in the pore6. In addition, improved bio-informatic tools like improvements in basecalling will improve the accuracy of basecalling7. One of the most frequently used basecallers, (e.g., Albacore), has been updated at least 12 times in a 9-month time period5. Recently, the manufacturer also released a novel basecaller called flip-flop, which is implemented in the default nanopore software8. Together, all of these improvements will lead to more accurate sequences and will decrease the error rate of the nanopore sequencer.
Usutu virus (USUV) is a mosquito-borne arbovirus of the family Flaviviridae and it has a positive-stranded RNA genome of around 11,000 nucleotides. USUV mainly affects great grey owls and blackbirds9,10, although other bird species are also susceptible to USUV infection11. Recently, USUV was also identified in rodents and shrews although their potential role in transmission of the virus remains unknown12. In humans, asymptomatic infections have been described in blood donors13,14,15,16 while USUV infections also have been reported to be associated with encephalitis or meningo-encephalitis17,18. In the Netherlands, USUV was first detected in wild birds in 201610 and in asymptomatic blood donors in 201814. Since the initial detection of USUV, outbreaks have been reported during the subsequent years and surveillance, including whole genome sequencing, is currently ongoing to monitor the emerge and spread of an arbovirus in a previously na&#239;ve population.
Similar to what has been described for other viruses, such as Ebola virus, Zika virus and yellow fever virus3,19,20, we have developed a primer set to sequence full length USUV21. This polymerase chain reaction (PCR)-based approach allows for the recovery of full length USUV genomes from highly host-contaminated sample types like brain samples in samples up to a Ct value of around 32. Benefits of an amplicon-based sequencing approach are a higher sensitivity compared to metagenomic sequencing and a higher specificity. Limitations of using an amplicon-based approach are that the sequences should be similar in order to design primers fitting all strains and that primers are designed on our current knowledge about the virus diversity.
Given the constant developments and improvements in third generation sequencing, there is a need to evaluate the error rate of the sequencer on a regular basis. Here, we describe a method to evaluate the performance of nanopore directly against Illumina sequencing using USUV as an example. This method is applied to sequences generated with the latest R10 flow cell and basecalling is performed with the latest version of the flip-flop basecaller.

<table><tbody><tr><td>Agencourt AMPure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>dNTPs</td><td>Qiagen</td><td>201900</td><td></td></tr><tr><td>FLO-MIN106 R10 flowcell</td><td>Nanopore</td><td>R10 flowcell</td><td></td></tr><tr><td>KAPA Hyperplus libarary preparation kit</td><td>Roche</td><td>7962436001</td><td></td></tr><tr><td>Library Loading Bead Kit</td><td>Nanopore</td><td>EXP-LLB001</td><td></td></tr><tr><td>Ligation Sequencing Kit 1D</td><td>Nanopore</td><td>SQK-LSK109</td><td></td></tr><tr><td>Native Barcoding Kit 1D 1-12</td><td>Nanopore</td><td>EXP-NBD103</td><td></td></tr><tr><td>Native Barcoding Kit 1D 13-24</td><td>Nanopore</td><td>EXP-NBD104</td><td></td></tr><tr><td>NEB Blunt/TA Ligase Master Mix</td><td>NEB</td><td>M0367S</td><td></td></tr><tr><td>NEB Next Quick Ligation Module</td><td>NEB</td><td>E6056</td><td></td></tr><tr><td>NEB Next Ultra II End Repair / dA-Tailing Module</td><td>NEB</td><td>E7546S</td><td></td></tr><tr><td>Protoscript II Reverse Transcriptase</td><td>NEB</td><td>M0368X</td><td></td></tr><tr><td>Q5 High-Fidelity polymerase</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>Qubit dsDNA HS Assay kit</td><td>Thermo Fisher</td><td>Q32851</td><td></td></tr><tr><td>Random Primers</td><td>Promega</td><td>C1181</td><td></td></tr><tr><td>RNAsin Ribonuclease Inhibitor</td><td>Promega</td><td>N2111</td><td></td></tr></tbody></table>

validating whole genome nanopore sequencing, using usutu virus as an example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

Recently, a new version of the flow cell version (R10) was released and offered improvements to the basecaller used to convert the electronic current signal to DNA sequences (so-called flip-flop basecaller). Therefore, we have re-sequenced USUV from brain tissue of an USUV-positive owl which was previously sequenced on a R9.4 flow cell and on an Illumina Miseq instrument21. Here, we described the method used to determine the required read coverage for reliable consensus calling by direct comparison to Illumina sequencing.
Using the newer flow cell in combination with the basecaller flip-flop we show that a read coverage of 40x results in identical results as compared to Illumina sequencing. A read coverage of 30x results in an error rate of 0.0002% which corresponds to one error in every 585,000 nucleotides sequenced, while a read coverage of 20x results in one error in every 63,529 nucleotides sequenced. A read coverage of 10x results in one error in every 3,312 nucleotides sequenced, meaning that over three nucleotides per full USUV genome are being called wrong. With a read coverage above 30x, no indels were observed. A read coverage of 20x resulted in the detection of one indel position while a read coverage of 10x resulted in indels in 29 positions. An overview of the error rate using different read coverage cut-offs is shown in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coverage</td>
			<td>Errors iteration 1</td>
			<td>Error rate iteration 1</td>
			<td>Indels:</td>
			<td>Errors iteration 2</td>
			<td>Error rate iteration 2</td>
			<td>Indels:</td>
			<td>Errors iteration 3</td>
			<td>Error rate iteration 3</td>
			<td>Indels:</td>
		</tr>
		<tr>
			<td>10&#215;</td>
			<td>100</td>
			<td>0.0274%</td>
			<td>4</td>
			<td>116</td>
			<td>0.0297%</td>
			<td>18</td>
			<td>110</td>
			<td>0.0282%</td>
			<td>7</td>
		</tr>
		<tr>
			<td>20&#215;</td>
			<td>4</td>
			<td>0.0010%</td>
			<td>0</td>
			<td>6</td>
			<td>0.0015%</td>
			<td>1</td>
			<td>7</td>
			<td>0.0018%</td>
			<td>0</td>
		</tr>
		<tr>
			<td>30x</td>
			<td>2</td>
			<td>0.0005%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
		</tr>
		<tr>
			<td>40x</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
		</tr>
		<tr>
			<td>50&#215;</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
			<td>0</td>
			<td>0.0000%</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of the error rate of nanopore sequencing. Each iteration represents one thousand random samples.
Supplementary File 1: Random selection. <a href="https://www.jove.com/files/ftp_upload/60906/Random_selection.zip" target="_blank">Please click here to view this file (Right click to download).</a>

Watch this Scientific Journal Video about Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example at JoVE.com

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

validating-whole-genome-nanopore-sequencing-using-usutu-virus-as-an

Research

JoVE Journal

Genetics

8.7K Views.  Erasmus University Medical Center. We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Video: Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Immunology and Infection

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

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

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

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, ...

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Medicine

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...