This work was funded by Genome BC, Genome Canada, and Coastal Genomics. The authors thank Kirby Cronin and Michael Chan for their help in sample collection and processing. We also would like to acknowledge Thea Van Rossum and Dr. Fiona Brinkman for bioinformatics assistance.

1. Coleta de Água e Filtração <ol><li> Coletar amostras de água doce de vários sites (Figura 1). Passe amostras através de uma série de filtros: filtro de 1 um, 0,2 um filtro, e de 30 kDa de corte de filtro de fluxo tangencial para sistematicamente eucariótica separada, partículas de tamanho bacterianas e virais, respectivamente. Nota: Apenas a purificação e análise do material recuperado nos filtros de 0,2 um (bactérias de vida livre) é descrito neste relatório, mas estratégias semelhantes podem ser usadas para outras partículas recuperadas a partir dos filtros. </li></ol> 2. Concentração bacteriana, ADN Extracção e Purificação <ol><li> Remover o filtro de membrana de 0,2 um (s) a partir do sistema de filtração de água (diagrama esquemático, Figura 2). Dobre o filtro de 0,2 um disco ao meio e coloque-o em um tubo de 50 ml com o lado aberto para cima. Adicionar 5 ml de solução tamponada com fosfato (PBS 1x) e 0,01% de Tween, pH 7,4 emo tubo. Se mais do que um filtro é utilizado durante o processo de empregar um tubo diferente por filtro. Tubo (s) de loja com filtro a 4 ° C até serem processadas. Caso contrário, vá para o passo 2.2. </li><li> Remover o filtro de membrana de 0,2 um a partir do tubo de 50 ml com uma pinça estéril. Ponto de filtro (s) em uma placa de Petri (deixar o tampão PBS no tubo). <ol><li> Com uma pinça estéril e tesoura estéril, cortou o filtro em pequenas tiras (1 cm x 1 cm). Desinfetar pinça e tesouras por imersão em 70% isopropanol, limpando com uma superfície descontaminante DNA e enxaguar com água ultrapura do tipo 1 entre as diferentes amostras. </li></ol></li><li> Colocar o filtro de corte em tiras de volta para o mesmo tubo de 50 ml. Adicionar 10 ml de 1x PBS e 0,01% de Tween, pH 7,4 para o filtro, de modo que há um total de 15 mL de tampão no tubo de 50 ml. </li><li> Adicione 6 esferas de tungstênio limpo (3 mm de diâmetro) para cada tubo de 50 ml, cobrir o tubo com Parafilm e vortex vigorosamente durante 20 min (use 50 ml tubos adaptador vortex). Se multiple filtros são processados ​​a partir de uma amostra, e transferência da piscina todo o homogeneizado para um tubo limpo de 50 ml. Centrifugar os tubos a 3300 xg durante 15 min a 4 ° C. </li><li> Ressuspender o sedimento de células bacterianas e alíquotas (~ 1 ml) em aliquotas de 1,7 ml tubos de microcentrífuga. Note-se que haverá cinco microtubos por amostra. </li><li> Girar os tubos de microcentrífuga a 10000 xg durante 10 min, e remover a maior parte do sobrenadante para baixo de ~ 200 ul marca. </li><li> Armazenar as amostras a -80 ° C, ou prosseguir para extrair ADN bacteriano usando um kit de isolamento de ADN comercialmente disponíveis de acordo com as instruções do fabricante. Durante a utilização de bactérias de extracção de ADN PBS ou água como um controlo negativo de extracção, e E. coli como controlo positivo extracção. </li><li> Como o DNA de vários tubos estão a ser extraídos, piscina sub-alíquotas paralelas a partir de uma mesma amostra em um tubo de 2 ml. Em seguida, conduzir uma precipitação S / N usando uma solução composta por 0,1volumes de acetato de sódio 3M, 2 volumes de etanol a 100%, e 5 uL de 5 ug / ul acrilamida linear. Misturar bem e armazenar amostras a -80 ° C. </li><li> Centrifuga-se a velocidade máxima (17000 xg) durante 30 min a 4 ° C. Lavar pelete com 1 ml de etanol a 70% arrefecido com gelo, ar seco numa câmara de biosegurança para não mais do que 5 minutos e ressuspender o sedimento de ADN em 34 ul de 10 mM Tris-Cl, pH 8,5. Nota: acrilamida linear irá ajudar a visualizar sedimento de DNA após a precipitação S / N. </li><li> Determinar a quantidade de ADN e de qualidade utilizando uma alta sensibilidade do ensaio de quantificação de ácidos nucleicos fluorescente e um espectrofotómetro microvolume, respectivamente, seguindo as instruções do fabricante (ver o quadro de Materiais). Nota: Se várias amostras são preparadas na hora, um ensaio nucleico fluorescente ultrasensitive ácido para quantificar dsDNA e baseado em placa fluorometer / espectrofotômetro pode ser usado em seu lugar. </li></ol> 3. Preparação de ADN da biblioteca <ol><li&gt; Normalizar o ADN genómico bacteriano extraído de amostras de água a uma concentração de ~ 0,2 ng / mL. </li><li> Enzimaticamente fragmento de um nanograma de ADN bacteriano usando um método baseado em transposões (tagmentation). Adicionar sequências de adaptador e de índice para DNA fragmentado seguindo as instruções do fabricante. Objecto tagmented amostras a 12 ciclos de PCR, como indicado nas instruções do fabricante, no ponto em que os índices de sequenciação são incorporados. </li></ol> 4. Automated Gel Seleção Tamanho <ol><li> Ir padrão pós-PCR passo de limpeza descrito nas instruções do fabricante (Ver Tabela de Materiais). Tamanho selecione amostras pós-PCR diretamente usando uma plataforma automatizada de seleção de tamanho com base em gel. <ol><li> Adicionar 3,5 uL de tampão de carga para cada amostra. </li><li> Seguindo o protocolo do fabricante, carregar as amostras para a estação de trabalho electroforese, especificando um tamanho alvo-selecção de 500-800 pb, num extracto de 300 uldo volume de iões, utilizando um pré-moldado de 1,5% de agarose a cassete. </li><li> Concentra-se o material resultante em bruto (300 uL) para baixo para 25 ul por precipitação com etanol. Nota: Como alternativa, use a estação de trabalho de eletroforese para automatizar concentração para um maior número de amostras através de placa de filtro e tubo de vácuo. <ol><li> Precipitar o volume de eluição em bruto utilizando 0,1 volumes de acetato de sódio 3M, 2 volumes de etanol a 100% e 5 uL de 5 ug / ul acrilamida linear. Misture bem, coloque as amostras a -80 ° CO / N. Centrifugar as amostras a 17.000 xg durante 30 min. </li><li> Rejeitar sobrenadante e prosseguir a lavagem sedimento de DNA com 1 mL de etanol gelado a 70%. </li><li> Centrifugar as amostras novamente a 17.000 xg durante 30 min, os sobrenadantes descartar, ar seco, e finalmente ressuspender pelota de ADN em 25 ul de 10 mM Tris-Cl, pH 8,5. </li></ol></li></ol></li><li> (Opcional) Use 1 ml de tamanho bibliotecas de DNA selecionado com a plataforma de seleção de tamanho de gel automatizado para fazer um cincodiluição -fold usando uma solução tampão de Tris-EDTA, pH 8,0. Analisar bibliotecas utilizando um instrumento de eletroforese capilar baseada em chip para analisar a qualidade de dsDNA de acordo com as instruções do fabricante. </li></ol> 5. Biblioteca Normalização e Sequencing <ol><li> Prossiga para normalizar amostras utilizando contas de normalização de biblioteca (incluído no kit de preparação biblioteca baseada transposon), conforme descrito no guia de preparação do fabricante. <ol><li> Alternativamente, bibliotecas de dsDNA medir utilizando um ensaio de quantificação de ácido nucleico de alta sensibilidade fluorescente, seguido por agrupamento em proporções equimolares até atingir uma concentração final de 2 nM dsDNA. Avance para desnaturar bibliotecas utilizando 10 ul de bibliotecas reunidas e 10 ul de NaOH 0,2 N, incubar durante 5 min à TA. Diluir bibliotecas desnaturados utilizando tampão de hibridação (incluída com o kit de preparação à base de transposão) para um volume final de 1 ml e a concentração de 23:05. </li></ol></li><li> Amostra de cargas em uma plataforma de sequenciamento de próxima geração, seguindo os protocolos de seqüenciamento padrão do fabricante. </li></ol>

<ol>
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R., 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=Recovery,+purification,+and+cloning+of+high-molecular-weight+DNA+from+soil+microorganisms.">Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms.</a> Applied and environmental microbiology. 74, 3302-3305 (2008).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+high+molecular+weight+DNA+from+microbial+mats.">Extraction of high molecular weight DNA from microbial mats.</a> BioTechniques. 49, 631-640 (2010).</li><li>Tebbe, C. C., Vahjen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+humic+acids+and+DNA+extracted+directly+from+soil+in+detection+and+transformation+of+recombinant+DNA+from+bacteria+and+a+yeast.">Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.</a> Applied and environmental microbiology. 59, 2657-2665 (1993).</li><li>Solonenko, S. 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+platform+and+library+preparation+choices+impact+viral+metagenomes.">Sequencing platform and library preparation choices impact viral metagenomes.</a> BMC genomics. 14, 320 (2013).</li><li>Aird, D., 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=Analyzing+and+minimizing+PCR+amplification+bias+in+Illumina+sequencing+libraries.">Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.</a> Genome biology. 12, R18 (2011).</li><li>Quail, M. A., Swerdlow, H., Turner, D. 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=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current protocols in human genetics / editorial board, Jonathan L. Haines .. [et al.]. 18, 18 (2009).</li><li>Head, S. R., 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=Library+construction+for+next-generation+sequencing:+overviews+and+challenges.">Library construction for next-generation sequencing: overviews and challenges.</a> BioTechniques. 56, 61-64, 66, 68 (2014).</li><li>Dijk, E. L., Jaszczyszyn, Y., Thermes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+methods+for+next-generation+sequencing:+tone+down+the+bias.">Library preparation methods for next-generation sequencing: tone down the bias.</a> Experimental cell research. 322, 12-20 (2014).</li><li>Grunenwald, H., Baas, B., Caruccio, N., Syed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+high-throughput+library+preparation+for+next-generation+sequencing.">Rapid, high-throughput library preparation for next-generation sequencing.</a> Nature Methods. 7, (2010).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of visualized experiments. , (2012).</li><li>Quail, M. 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=A+large+genome+center's+improvements+to+the+Illumina+sequencing+system.">A large genome center's improvements to the Illumina sequencing system.</a> Nature methods. 5, 1005-1010 (2008).</li><li>Marine, R., 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=Evaluation+of+a+transposase+protocol+for+rapid+generation+of+shotgun+high-throughput+sequencing+libraries+from+nanogram+quantities+of+DNA.">Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.</a> Applied and environmental microbiology. 77, 8071-8079 (2011).</li><li>Rohland, N., Reich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+high-throughput+DNA+sequencing+libraries+for+multiplexed+target+capture.">Cost-effective high-throughput DNA sequencing libraries for multiplexed target capture.</a> Genome research. 22, 939-946 (2012).</li><li>Lamble, S., 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+workflows+for+high+throughput+library+preparation+using+the+transposome-based+nextera+system.">Improved workflows for high throughput library preparation using the transposome-based nextera system.</a> Bmc Biotechnology. 13, (2013).</li><li>Picelli, S., 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=Tn5+transposase+and+tagmentation+procedures+for+massively+scaled+sequencing+projects.">Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.</a> Genome research. , (2014).</li><li>Rhodes, J., Beale, M. 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Jared R. Slobodan e Matthew J. Nesbitt tanto deter ações da Coastal Genomics, uma empresa British Columbia propriedade privada oferecendo a tecnologia Ranger.

A presença de dímeros de adaptador e aglomeração de pequenas dimensões de inserção preferencialmente sendo sequenciados em plataformas atuais representam uma diminuição nos rendimentos utilizáveis ​​e subutilização da capacidade do equipamento 15. A utilização de métodos de batimento grânulos combinados com uma abordagem baseada no transposão para preparar bibliotecas podem resultar em mais de cisalhamento de ADN em comparação com os outros métodos de extracção de 4,5. No ...

Metagenomics involves the sequencing of all the genetic material in a sample to characterize the microbial communities present. It is a complex and expensive process which involves the conversion of extracted nucleic acids into DNA libraries followed by next-generation sequencing. High quality libraries are essential for maximal data output and accurate metagenomics analysis. Environmental samples, such as water samples, often pose significant challenges to generating high quality libraries, due to low amounts of DNA that may also be degraded1-3 and the presence of inhibitors of PCR4-6. 
High quality libraries ideally consist of longer segments of DNA within a narrow range of lengths. In order to maximize the amount of useful data generated per sequencing run, the length of the DNA in the library should be at least as long as the maximum read length of the sequencing method being used. When using a sequencing-by-synthesis technology such as the Illumina MiSeq, the size of the DNA fragments affects the efficiency at which clusters are generated on the flow cell. For instance, when a library contains both shorter and longer DNA fragments, the shorter ones will be over-represented in the sequencing data7,8. In contrast, a library with similarly sized DNA fragments will be proportionally represented in the sequencing data. Many library preparation kits use ligation-based methods to add adapters to the DNA fragments and size selection is necessary to remove adapter dimers that do not contain an insert9,10. There are numerous methods11,12 to achieve this but the one technique that gives the most consistent results is the electrophoretic separation of DNA followed by the recovery of the desired lengths of DNA13,14. This process can be performed manually for a small number of samples, but when faced with processing hundreds of samples, automated solutions are required. The currently available platforms for automated gel size selection are low throughput and new platforms are needed to process large numbers of samples for sequencing. The Ranger Technology can be integrated with existing liquid handling workstations to enable the use of agarose gel electrophoresis for size selection and analytical purposes on a scale that satisfies today&#8217;s high throughput environment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Peristaltic pump Masterflex P/S 1400 Series</td>
			<td>Thermo Scientific&#160;</td>
			<td>1400-1620</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m Supor Membrane</td>
			<td>VWR</td>
			<td>CA28143-969</td>
			<td>Pall Corporation, Ann Harbor, MI</td>
		</tr>
		<tr>
			<td>Tungsten carbide beads 3 mm (200)</td>
			<td>Qiagen</td>
			<td>69997</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol 70%</td>
			<td>Jedmon Products</td>
			<td>825751</td>
			<td>Healthcare Plus</td>
		</tr>
		<tr>
			<td>DNA away</td>
			<td>VWR</td>
			<td>7010</td>
			<td>Molecular BioProducts, Inc. San Diego, CA</td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>Fisher Scientific</td>
			<td>ZMQS6VF0Y</td>
			<td>Merck Millipore. This system has been discontinued.</td>
		</tr>
		<tr>
			<td>20 X PBS pH 7.5</td>
			<td>VWR</td>
			<td>E703-1L</td>
			<td>Amresco, Inc., Solon, OH</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td>Fisher chemicals</td>
		</tr>
		<tr>
			<td>Vortex adapter for 2 (50 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-50</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2, 120V (Model G560)</td>
			<td>VWR</td>
			<td>SI-0236</td>
			<td>Scientific Industries, Inc.</td>
		</tr>
		<tr>
			<td>Beckman Centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd</td>
			<td>Model J-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer Powersoil DNA isolation kit</td>
			<td>VWR</td>
			<td>12855-100</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex adapter for 24 (1.5-2 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-24</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Microfuge 18 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>367160</td>
			<td></td>
		</tr>
		<tr>
			<td>Nimbus Select workstation with Ranger Technology</td>
			<td>Hamilton Robotics</td>
			<td>92720-01</td>
			<td>Includes the liquid handling workstation and integrated Ranger Tech (electrophoresis hardware)</td>
		</tr>
		<tr>
			<td>Ranger reagent kit</td>
			<td>Coastal Genomics</td>
			<td>CG-10600-150-12-21</td>
			<td>Includes loading buffer and cassettes</td>
		</tr>
		<tr>
			<td>Ethyl alcohol (anhydrous)</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td>Greenfield Ethanol</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear acrylamide (5 mg/ml)</td>
			<td>Life Technologies</td>
			<td>AM9520</td>
			<td>Ambion</td>
		</tr>
		<tr>
			<td>Eppendorf refrigerated centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd.</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer EB (250 ml)</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32857</td>
			<td>Invitrogen. This product has been discontinued.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Life Technologies</td>
			<td>Q32854</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>High sensitivity DNA reagent</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity DNA chips</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2011 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA sample preparation kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT index kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>MiSeq reagent kit v2 (500-cycles)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Miseq system</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated gel size selection to improve the quality of next-generation sequencing libraries prepared from environmental water samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

Concentração DNA, e Avaliação da Qualidade As concentrações de DNA bacterianas, variou de 0,01-0,11 ng ​​/ ml por amostra de água dos diferentes sítios de bacias (Tabela 1). O ADN isolado a partir de uma fracção bacteriana teve 260/280 e 260/230 A rácios de 1,4 a 1,8 e 0,3 a 1,6, respectivamente. Enquanto alguns dos rácios Um 260/230 foram relativamente baixos, não foi observada inibição aparente na bibliot...

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Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Automated Gel seleção de tamanho para melhorar a qualidade das bibliotecas de sequenciação de próxima geração Preparado a partir de amostras de água do ambiente

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

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10.4K visualizações.  The University of British Columbia. This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries. 

Vídeo: Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Método 1615. Medição de Enterovirus e Norovirus Ocorrência na água pela cultura e RT-qPCR. I. Recolha de amostras de vírus

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Ambiente

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

O isolamento físico do Endospores de amostras ambientais por alvejado Lise de células vegetativas

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Um método simples para a extração automatizada de fase sólida das amostras de água para análise imunológica de Pequenas Poluentes

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

O uso de um cartucho de filtro para Amostras de filtração de água e extração de DNA Ambiental

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties of the pouch were monitored in containers with or without fruit. The pouch was affixed to the inside of a perforated clamshell containing grape tomatoes, and the effect on microbial population, firmness, and weight loss was evaluated during a 14 day storage period at 20 &#176;C. Within 3 days, the ClO2 concentration in the clamshells reached 3.5 ppm and remained constant until day 10. Thereafter, it decreased to 2 ppm by day 14. The ClO2 pouch exhibited strong antimicrobial activity, reducing Escherichia coli populations by 3.08 log CFU/g and Alternaria alternata populations by 2.85 log CFU/g after 14 days of storage. The ClO2 treatment also reduced softening and weight loss and extended the overall shelf life of the tomatoes. Our results suggest that ClO2 treatment is useful for extending the shelf life and improving the microbial safety of tomatoes during storage without impairing their quality.

Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

A libertação controlada de dióxido de cloro em um sistema de embalagem perfurada para prolongar a vida de armazenamento e melhorar a segurança dos Tomates da uva

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extração de Pesticidas Organoclorados de Pelotização de Plástico e Análise de Tipo de Plástico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Instalação de coluna experimental para estudar anaeróbicas biogeoquímicas interações entre bactérias, oligoelementos e hidróxidos de ferro (oxi)

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

Contínua hidrológicos e monitoramento da qualidade do Vernal lagoas de água

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

Procedimento otimizado para determinar a adsorção de fosfonatos para Granular hidróxido férrico, usando um método de determinação de fósforo miniaturizado

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Composição e análise da distribuição de Bioaerossóis sob diferentes condições ambientais

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...