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Joran Martijn Anders E. Lind Max E. Schön Ian Spiertz Lina Juzokaite Ignas Bunikis Olga V. Pettersson Thijs J. G. Ettema 《Environmental microbiology》2019,21(7):2485-2498
Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. 相似文献
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Lina Long Douglas R. McCabe M. Eileen Dolan 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,731(2):128
A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C18 reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O6-benzylguanine in a phase I clinical trial. Previously, O6-benzyl-8-oxoguanine was identified as the primary metabolite of O6-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O6-benzylguanine. 相似文献
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Ross A. Robinson Samuel C. Griffiths Lieke L. van de Haar Tomas Malinauskas Eljo Y. van Battum Pavol Zelina Rebekka A. Schwab Dimple Karia Lina Malinauskaite Sara Brignani Marleen H. van den Munkhof Özge Düdükcü Anna A. De Ruiter Dianne M.A. Van den Heuvel Benjamin Bishop Jonathan Elegheert A. Radu Aricescu R. Jeroen Pasterkamp Christian Siebold 《Cell》2021,184(8):2103-2120.e31
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Xuan He Abishek Chandrashekar Roland Zahn Frank Wegmann Jingyou Yu Noe B. Mercado Katherine McMahan Amanda J. Martinot Cesar Piedra-Mora Sidney Beecy Sarah Ducat Ronnie Chamanza Sietske Rosendahl Huber Marjolein van Heerden Leslie van der Fits Erica N. Borducchi Michelle Lifton Jinyan Liu Dan H. Barouch 《Cell》2021,184(13):3467-3473.e11
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