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Authors: Benjamin Istace, Anne Friedrich, Léo d’Agata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury
Group: GigaScience Press, High molecular weight DNA extraction from all kingdoms
Summary: These protocols accompany the following GigaScience publication:Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. GigaScience...
Proper citation: Benjamin Istace, Anne Friedrich, Léo d’Agata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury 2017. Protocols from "De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer". protocols.io dx.doi.org/10.17504/protocols.io.gvxbw7n Copy
Authors: Chen Chen, Lilan Hao, Weixia Wei, Fei Li, Liju Song, Xiaowei Zhang, Juanjuan Dai, Zhuye Jie, Jiandong Li, Xiaolei Song, Zirong Wang, Zhe Zhang, Liping Zeng, Hui Du, Huiru Tang, Tao Zhang, Huanming Yang, Jian Wang, Susanne Brix, Karsten Kristiansen, Xun Xu, Ruifang Wu, Huijue Jia
Group: GigaScience Press, BGI
Summary: Human urine is traditionally considered to be sterile, and whether the urine harbours distinct microbial communities has been a matter of debate. Potential links between female urine and reproductive tract microbial communities is currently not clear. Here, we collected urine samples from 147 Chinese women of reproductive age and explored the nature of colonization by 16S rRNA gene amplicon sequencing, quantitative real-time PCR, and live bacteria culture. To demonstrate the utility of this approach, the intra-individual Spearman's correlation was used to explore the relationship between urine and multiple sites of the reproductive tract. PERMANOVA was also performed to explore potential correlations between the lifestyle and various clinical factors and urinary bacterial communities. Our data demonstrated distinct bacterial communities in urine, indicative of a non-sterile environment. Streptococcus-dominated, Lactobacillus-dominated, and diverse type were the three most common urinary bacterial community types in the cohort. Detailed comparison of the urinary microbiota with multiple sites of the female reproductive tract microbiota demonstrated that the urinary microbiota were more similar to the microbiota in the cervix and uterine cavity than to those of the vagina in the same women. Our data demonstrate the potential connectivity among microbiota in the female urogenital system and provide insight and resources for exploring diseases of the urethra and genital tract.
Proper citation: Chen Chen, Lilan Hao, Weixia Wei, Fei Li, Liju Song, Xiaowei Zhang, Juanjuan Dai, Zhuye Jie, Jiandong Li, Xiaolei Song, Zirong Wang, Zhe Zhang, Liping Zeng, Hui Du, Huiru Tang, Tao Zhang, Huanming Yang, Jian Wang, Susanne Brix, Karsten Kristiansen, Xun Xu, Ruifang Wu, Huijue Jia 2020. Protocols for "The female urinary microbiota in relation to the reproductive tract microbiota.". protocols.io dx.doi.org/10.17504/protocols.io.bp3wmqpe Copy
Authors: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong
Group: GigaScience Press, BGI
Summary: The 1000 Plants (1KP) initiative explored the genetic diversity of green plants (Viridiplantae) by sequencing RNA from 1,342 samples representing 1,173 species. All of the analyses done for the 1KP capstone, and previous studies on subsets of these data, are based on a series of de novo transcriptome assemblies and related outputs that will be described in this publication. We expect that these data will also be useful to other researchers with interests in specific gene families, either across the green plant tree of life or in more focused lineages. These protocols provide additional details on the sample and sequence processing, hopefully providing insight for reproducibility and tools for other researchers working on plant transcriptomics and evolution.
Proper citation: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong 2019. Sequencing Protocols for the One Thousand Plant Transcriptomes Initiative. protocols.io dx.doi.org/10.17504/protocols.io.38jgrun Copy
Authors: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma
Group: GigaScience Press
Summary: Long Mate Pair Library Construction Protocol
Proper citation: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma 2020. Long Mate Pair Library Construction Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bd3ui8nw Copy
Authors: Eric Carpenter
Group: GigaScience Press
Summary: Implemented by: Michael Melkonian and Barbara SurekThe protocol for RNA isolation using TRI Reagent (Molecular Research Center, Cincinnati OH) is analogous to the TRIzol LS Reagent protocol except that 1 ml of TRI Reagent is added to each 250 μl of homogenized material (= approx. 50–100 mg packed cell volume) (step 3, Protocol 10). For background, TRI Reagent combines phenol and guanidine thiocyanate in a monophasic solution to inhibit RNase activity during RNA isolation.This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6)
Proper citation: Eric Carpenter 2019. RNA Isolation from Plant Tissue Protocol 11: Tri Reagent Method. protocols.io dx.doi.org/10.17504/protocols.io.4ydgxs6 Copy
Authors: Marc T. J. Johnson, Eric J. Carpenter, Zhijian Tian, Richard Bruskiewich, Jason N. Burris, Charlotte T. Carrigan, Mark W. Chase, Neil D. Clarke, Sarah Covshoff, Claude W. dePamphilis, Patrick P. Edger, Falicia Goh, Sean Graham, Stephan Greiner, Julian M. Hibberd, Ingrid Jordon-Thaden, Toni M. Kutchan, James Leebens-Mack, Michael Melkonian, Nicholas Miles, Henrietta Myburg, Jordan Patterson, J. Chris Pires, Paula Ralph, Megan Rolf, Rowan F. Sage, Douglas Soltis, Pamela Soltis, Dennis Stevenson, C. Neal Stewart Jr, Barbara Surek, Christina J. M. Thomsen, Juan Carlos Villarreal, Xiaolei Wu, Yong Zhang, Michael K. Deyholos, Gane Ka-Shu Wong
Group: GigaScience Press
Summary: Methods for RNA isolationThese methods were originally included in Appendix S1 of "Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes" Marc T. J. Johnson et al. PLOS ONE, November 21, 2012. https://doi.org/10.1371/journal.pone.0050226Many of the protocols share elements or combine components from several methods. For each method, we describe the reagents and procedures used, and identify the researchers or institute that implemented the protocol.Due to the potential for contamination and degradation by RNase enzymes, as well as health concerns in handling some substances and chemicals, best practices in aseptic wet lab techniques must be practiced at all times during RNA isolation. Chief among these are the critical need to avoid contamination of samples by using extreme care when moving liquids and opening and closing tubes to avoid aerosols. Because of the risk of degradation by RNase enzymes, it is essential to use sterile RNase-free equipment, disposable plastics and solutions.RNase degradation and contamination can be avoided by keeping samples constantly frozen at low temperature (1. Many additional helpful tips for successful RNA isolation are available in Sambrook and Russell1 and in Appendix A of Qiagen’s RNeasy Mini Handbook downloadable from www.qiagen.com.1 Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, 2001).
Proper citation: Marc T. J. Johnson, Eric J. Carpenter, Zhijian Tian, Richard Bruskiewich, Jason N. Burris, Charlotte T. Carrigan, Mark W. Chase, Neil D. Clarke, Sarah Covshoff, Claude W. dePamphilis, Patrick P. Edger, Falicia Goh, Sean Graham, Stephan Greiner, Julian M. Hibberd, Ingrid Jordon-Thaden, Toni M. Kutchan, James Leebens-Mack, Michael Melkonian, Nicholas Miles, Henrietta Myburg, Jordan Patterson, J. Chris Pires, Paula Ralph, Megan Rolf, Rowan F. Sage, Douglas Soltis, Pamela Soltis, Dennis Stevenson, C. Neal Stewart Jr, Barbara Surek, Christina J. M. Thomsen, Juan Carlos Villarreal, Xiaolei Wu, Yong Zhang, Michael K. Deyholos, Gane Ka-Shu Wong 2019. RNA Isolation from Plant Tissue. protocols.io dx.doi.org/10.17504/protocols.io.439gyr6 Copy
Authors: Tianlin Pei, Mengxiao Yan, Yu Kong, Hang Fan, Jie Liu, Mengying Cui, Yumin Fang, Binjie Ge, Jun Yang, Qing Zhao
Group: BGI, GIGA, GigaScience Press
Summary: Tripterygium wilfordii is a vine from the Celastraceae family that is used in traditional Chinese medicine (TCM). The active ingredient celastrol is a friedelane-type pentacyclic triterpenoid with putative roles as antitumor, immunosuppressive, and antiobesity agent. Here, we report a reference genome assembly of T. wilfordii with high-quality annotation by using a hybrid sequencing strategy, obtaining a 340.12 Mb total genome size, a contig N50 reaching 3.09 Mb. We successfully anchored 91.02% of the sequences into 23 pseudochromosomes using Hi-C technology and the superscaffold N50 reached 13.03 Mb. In addition, we annotated 31,593 structural genes, and a repeat percentage of 44.31%. Comparative evolutionary analyses showed that T. wilfordii diverged from species of Malpighiales approximately 102.4 million years ago. Based on integration of the genome, transcriptome and metabolite analyses, as well as in vivo and in vitro enzyme assays of the two CYP450 genes TwCYP712K1 and TwCYP712K2 the second biosynthesis step of celastrol was investigated. Syntenic analysis revealed that TwCYP712K1 and TwCYP712K2 were derived from a common ancestor. These results provide insights for further investigating pathways related to celastrol andvaluable information to aid the conservation of resources. In addition, they helped us understand the evolution of Celastrales.
Proper citation: Tianlin Pei, Mengxiao Yan, Yu Kong, Hang Fan, Jie Liu, Mengying Cui, Yumin Fang, Binjie Ge, Jun Yang, Qing Zhao 2021. Protocols for "The genome analysis of Tripterygium wilfordii reveals TwCYP712K1 and TwCYP712K2 responsible for oxidation of friedelin in celastrol biosynthesis pathway". protocols.io dx.doi.org/10.17504/protocols.io.bspfndjn Copy
Authors: Sarah Siu Tze Mak, Shyam Gopalakrishnan, Christian Caroe, Chunyu Geng, Shanlin Liu, Mikkel-Holger S Sinding, Lukas F K Kuderna, Wenwei Zhang, Shujin Fu, Filipe G Vieira, Mietje Germonpré, Hervé Bocherens, Sergey Fedorov, Bent Petersen, Thomas Sicheritz-Ponten, Tomas Marques-Bonet, Guojie Zhang, Hui Jiang, M Thomas P Gilbert
Group: GigaScience Press
Summary: Ancient DNA research has been revolutionized following the development of ‘Next Generation’ Sequencing platforms. Although a number of such platforms have been applied to ancient DNA samples, the Illumina series are the dominant choice today, mainly because of high production capacities and short read production. Recently a potentially attractive alternative platform for palaeogenomic data generation has been developed, the BGISEQ-500, whose sequence output are comparable with the Illumina series. In this study, we modified the standard BGISEQ-500 library preparation specifically for use on degraded DNA, then directly compared the sequencing performance and data quality of the BGISEQ-500 to the Illumina HiSeq2500 platform, on DNA extracted from eight historic and ancient dog and wolf samples. The data generated was largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (p=0.371) and average sequence length (p=0718) of endogenous nuclear DNA, sequence GC content (p=0.311), double-stranded DNA damage rate (p=0.309), and sequence clonality (p=0.093). Small significant differences were found in single strand DNA damage rate (δS, slightly lower for the BGISEQ-500, p=0.011) and the background rate of difference from the reference genome (θ, slightly higher for BGISEQ-500, p=0.012). This may result from the differences in amplification cycles used to PCR amplify the libraries. A significant difference was also observed in the mitochondrial DNA percentages recovered (p=0.018), although we believe this is likely a stochastic effect relating to the extremely low levels of mitochondria that were sequenced from three of the samples with overall very low levels of endogenous DNA. Although we acknowledge our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent valid and potentially valuable alternative platform for palaeogenomic data generation, that is worthy of future exploration by those interested in the sequencing and analysis of degraded DNA.
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Proper citation: Sarah Siu Tze Mak, Shyam Gopalakrishnan, Christian Caroe, Chunyu Geng, Shanlin Liu, Mikkel-Holger S Sinding, Lukas F K Kuderna, Wenwei Zhang, Shujin Fu, Filipe G Vieira, Mietje Germonpré, Hervé Bocherens, Sergey Fedorov, Bent Petersen, Thomas Sicheritz-Ponten, Tomas Marques-Bonet, Guojie Zhang, Hui Jiang, M Thomas P Gilbert 2017. Protocols from "Comparative performance of the BGISEQ-500 vs Illumina sequencing platforms for palaeogenomic sequencing". protocols.io dx.doi.org/10.17504/protocols.io.h99b996 Copy
Authors: Rui Zhang
Group: BGI, GIGA, GigaScience Press
Summary: The humpback puffer,Tetraodon palembangensis, is a species of poisonous freshwater pufferfish mainly distributed in Southeast Asia (Thailand, Laos, Malaysia and Indonesia). The humpback puffer has many interesting biological features, such as inactivity, tetrodotoxin production and body expansion. Here, we reported the first chromosome-level genome assembly of the humpback puffer. The genome size is 362 Mb with ~1.78 Mb contig N50 and ~15.8 Mb scaffold N50. Based on the genome, ~61.5Mb (18.11%) repeat sequences were identified, 19,925 genes were annotated, and 90.01% of these genes could be predicted with function. Finally, a phylogenetic tree of ten teleost fish species was constructed, which suggests that humpback puffer and T. nigroviridis shared a common ancestor at 18.1 MYA and diverged from T. rubripes at 45.8 MYA. The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance.
Proper citation: Rui Zhang 2021. Oxford Nanopore sequencing and library construction. protocols.io dx.doi.org/10.17504/protocols.io.btcwnixe Copy
Authors: Yasmin Bar El
Group: GigaScience Press
Summary: Isolated astrocyte cell culture preparation. Dissociation, preparation and plating of mice cortex neurons and glia cells on MEA. Isolatioin process of the culture from neurons, to obtain an isolated astrocyte culture.
Proper citation: Yasmin Bar El 2018. Isolated astrocyte culture preparation - protocol 1. protocols.io dx.doi.org/10.17504/protocols.io.tqbemsn Copy
Authors: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma
Group: GigaScience Press
Summary: 10x Genomics Library Construction
Proper citation: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma 2020. 10x Genomics Library Construction. protocols.io dx.doi.org/10.17504/protocols.io.bd3vi8n6 Copy
Authors: Faezah Mohd Salleh, Jazmin Ramos-Madrigal, Fernando Penaloza, Shanlin Liu, Mikkel-Holger S Sinding, Riddhi P Patel, Renata Martins, Dorina Lenz, Jorns Fickel, Christian Roos, Mohd Shahir Shamsir, Mohammad Shahfiz Azman, Burton K Lim, Stephen J Rossiter, Andreas Wilting, M Thomas P Gilbert
Group: GigaScience Press
Summary: This protocol provides an efficient DNA extraction and purification of historical museum hides, which potentially have been chemically tanned.
Proper citation: Faezah Mohd Salleh, Jazmin Ramos-Madrigal, Fernando Penaloza, Shanlin Liu, Mikkel-Holger S Sinding, Riddhi P Patel, Renata Martins, Dorina Lenz, Jorns Fickel, Christian Roos, Mohd Shahir Shamsir, Mohammad Shahfiz Azman, Burton K Lim, Stephen J Rossiter, Andreas Wilting, M Thomas P Gilbert 2017. Extraction method C (FMS). protocols.io dx.doi.org/10.17504/protocols.io.inbcdan Copy
Authors: Chang Li
Group: GigaScience Press, BGI
Summary: From this protocol, we can know detail methods of assembly and annotation of the L. maculatus genome.
Proper citation: Chang Li 2018. Fish genome assembly and annotation pipeline. protocols.io dx.doi.org/10.17504/protocols.io.ss3eegn Copy
Authors: Robert Auber
Group: Wisecaver Lab, BGI, GIGA, GigaScience Press
Proper citation: Robert Auber 2019. Total DNA extraction from plant tissue using CTAB method. protocols.io dx.doi.org/10.17504/protocols.io.bamnic5e Copy
Authors: Chang Li
Group: GigaScience Press, BGI
Summary: This protocol is used to clarity the process of the short insert size WGS libraries preparation for the L. maculatus.
Proper citation: Chang Li 2018. Short insert size WGS libraries preparation for assembly of the Lateolabrax maculatus genome. protocols.io dx.doi.org/10.17504/protocols.io.sszeef6 Copy
Authors: Chang-Ming Bai
Group: BGI, GIGA, GigaScience Press
Summary: This protocol include the detailed methods of Hi-C assembly of the Scapharca broughtonii genome
Proper citation: Chang-Ming Bai 2019. The pipeline of Hi-C assembly of the Scapharca broughtonii genome. protocols.io dx.doi.org/10.17504/protocols.io.z8cf9sw Copy
Authors: Guangyi Fan, Judy Chan, Kailong Ma, Simon Ming-Yuen Lee, Binrui Yang, He Zhang, Xianwei Yang, Chengcheng Shi, Henry Law, Zhitao Ren, Qiwu Xu, Qun Liu, Jiahao Wang, Wenbin Chen, Libin Shao, David Gonçalves, Andreia Ramos, Sara D. Cardoso, Min Guo, Jing Cai, Xun Xu, Jian Wang, Huanming Yang, Xin Liu, Yitao Wang
Group: GigaScience Press, BGI
Summary: Siamese fighting fish Betta splendens are notorious for their aggressiveness and accordingly have been widely used to study aggression. However, the lack of a reference genome has so far limited the understanding of the genetic basis of aggression in this species. Here we present the first reference genome assembly of the Siamese fighting fish.We first sequenced and de novo assembled a 465.24 Mb genome for the B. splendens variety Giant, with a weighted average (N50) scaffold size of 949.03 Kb and an N50 contig size of 19.01 Kb, covering 99.93% of the estimated genome size. To obtain a chromosome-level genome assembly, we constructed one Hi-C library and sequenced 75.24 Gb reads using the BGISEQ-500 platform. We anchored approximately 93% of the scaffold sequences into 21 chromosomes and evaluated the quality of our assembly using the high contact frequency heatmap and BUSCO. We also performed comparative chromosome analyses between Oryzias latipes and B. splendens, revealing a chromosome conservation evolution in B. splendens. We predicted a total of 23,981 genes assisted by RNA-seq data generated from brain, liver, muscle and heart tissues of Giant, and annotated 15% repetitive sequences in the genome. Additionally, we resequenced other five B. splendens varieties and detected ~3.4M single-nucleotide variations (SNVs) and 27,305 indels.We provide the first chromosome-level genome for the Siamese fighting fish. The genome will lay a valuable foundation for future research on aggression in B. splendens.
Proper citation: Guangyi Fan, Judy Chan, Kailong Ma, Simon Ming-Yuen Lee, Binrui Yang, He Zhang, Xianwei Yang, Chengcheng Shi, Henry Law, Zhitao Ren, Qiwu Xu, Qun Liu, Jiahao Wang, Wenbin Chen, Libin Shao, David Gonçalves, Andreia Ramos, Sara D. Cardoso, Min Guo, Jing Cai, Xun Xu, Jian Wang, Huanming Yang, Xin Liu, Yitao Wang 2018. High-quality reference genome of the Siamese fighting fish Betta splendens, a model species for the study of aggression. protocols.io dx.doi.org/10.17504/protocols.io.qvddw26 Copy
Authors: Qi Wang
Group: BGI, GIGA, GigaScience Press
Summary: The calculation of gut metabolic modules from gene profile
Proper citation: Qi Wang 2021. The calculation of gut metabolic modules from gene profile. protocols.io dx.doi.org/10.17504/protocols.io.bq6gmzbw Copy
Authors: Eric Carpenter
Group: GigaScience Press
Summary: Implemented by: Ingrid Jordon-Thaden and Nicholas Miles (Soltis Labs)This procedure eliminates the mortar and pestle homogenization of tissues and instead grinds tissue in 2 ml microcentrifuge tubes. The method closely follows Ambion’s protocols and could be used in a 96-well format. This method worked great for species that proved to be difficult to extract with other methods (i.e. woody and aquatic plants).This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6)
Proper citation: Eric Carpenter 2019. RNA Isolation from Plant Tissue Protocol 14: Ambion Trizol RNA Extraction in Microcentrifuge Tubes with Turbo DNAfree Digestion. protocols.io dx.doi.org/10.17504/protocols.io.4u6gwze Copy
Authors: David O'Connor, Natan Vega Potler, Meagan Kovacs, Ting Xu, Lei Ai, John Pellman, Tamara Vanderwal, Lucas Parra, Samantha Cohen, Satrajit Ghosh, Jasmine Escalera, Natalie Grant-Villegas, Yael Osman, Anastasia Bui, R Cameron Craddock, Michael P Milham
Group: GigaScience Press
Summary: These protocols accompany the following publication:David O'Connor, et. al. (2017) The Healthy Brain Network Serial Scanning Initiative. GigaScience...
Proper citation: David O'Connor, Natan Vega Potler, Meagan Kovacs, Ting Xu, Lei Ai, John Pellman, Tamara Vanderwal, Lucas Parra, Samantha Cohen, Satrajit Ghosh, Jasmine Escalera, Natalie Grant-Villegas, Yael Osman, Anastasia Bui, R Cameron Craddock, Michael P Milham 2017. Protocols for The Healthy Brain Network Serial Scanning Initiative (Sessions 1-14). protocols.io dx.doi.org/10.17504/protocols.io.gxcbxiw Copy
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