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Authors: Rui Zhang
Group: BGI, GIGA, GigaScience Press
Summary: This is a protocol for Oxford Nanopore sequencing and library construction, which was used in the humpback puffer genome sequence.

Proper citation: Rui Zhang 2021. Oxford Nanopore sequencing and library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.btignkbw Copy   

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Authors: Changwei Shao, Chang Li, Na Wang, Yating Qin, Wenteng Xu, Qun Liu, Qian Zhou, Yong Zhao, Xihong Li, Shanshan Liu, Xiaowu Chen, Shahid Mahboob, Xin Liu, Songlin Chen
Group: GigaScience Press, BGI
Summary: Background: The spotted sea bass (Lateolabrax maculatus) is a valuable commercial fish that is widely cultured in China. While analyses using molecular markers and population genetics have been conducted, genomic resources are lacking.Finding: Here, we report a chromosome-scale assembly of the spotted sea bass genome by high-depth genome sequencing, assembly and annotation. The genome scale was 0.62 Gb with contig and scaffold N50 length to be 31 Kb and 1,040 Kb, respectively. Hi-C scaffolding of the genome resulted in 24 pseudochromosomes containing 77.68% of the total assembled sequences. A total of 132.38Mb repeat sequences were detected, accounting for 20.73% of the assemble genome. 22,015 protein-coding genes were predicted, of which 96.52% were homologous to proteins in various databases. In addition, we constructed a phylogenetic tree using 1,586 single-copy gene families and identified 125 unique gene families in the spotted sea bass genome.Conclusions: We assembled a spotted sea bass genome, which will be a valuable genomic resource to understand the biology of the spotted sea bass, and will also lead to the development of molecular breeding techniques to generate spotted sea bass with better economical traits.

Proper citation: Changwei Shao, Chang Li, Na Wang, Yating Qin, Wenteng Xu, Qun Liu, Qian Zhou, Yong Zhao, Xihong Li, Shanshan Liu, Xiaowu Chen, Shahid Mahboob, Xin Liu, Songlin Chen 2018. Protocols for chromosome-level genome assembly of the spotted sea bass, Lateolabrax maculatus. protocols.io https://dx.doi.org/10.17504/protocols.io.ssxeefn Copy   

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Authors: Yasmin Bar El
Group: GigaScience Press
Summary: Protocols for Primary cortical neuronal-astrocyte cell culture, Isolated astrocyte cell culture, Immunocytochemistry, Electrophysiology and Ca2+ imaging.

Proper citation: Yasmin Bar El 2018. Protocols for activity changes in Neuron-Astrocyte Networks in Culture Under the Effect of Norepinephrine. protocols.io https://dx.doi.org/10.17504/protocols.io.tp9emr6 Copy   

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Authors: Eric Carpenter
Group: GigaScience Press
Summary: Implemented by: Michael Melkonian and Barbara Surek (algae) and Juan Carlos Villarreal (bryophytes)This protocol follows the procedures provided with the TRIzol LS Reagent (Invitrogen). TRIzol LS Reagent is a monophasic solution of phenol and guanidine isothiocyanate that can be used in isolation of total RNA from a wide variety of tissues and organisms, in addition to plants. This protocol was used in the isolation of total RNA from some algae samples (see Supplementary Table 1).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 10: TRIzol LS Reagent Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4rwgv7e Copy   

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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: Whilst much sequencing effort has focused on key mammalian model organisms such as mouse and human, little is known about the correlation between genome sequencing techniques for non-model mammals and genome assembly quality. This is especially relevant to non-model mammals, where the samples to be sequenced are often degraded and low quality. A key aspect when planning a genome project is the choice of sequencing data to generate. This decision is driven by several factors, including the biological questions being asked, the quality of DNA available, and the availability of funds. Cutting-edge sequencing technologies now make it possible to achieve highly contiguous, chromosome-level genome assemblies, but relies on good quality high-molecular-weight DNA. Here we use a range of different genomic technologies generated from a roadkill European Polecat (Mustela putorius) to assess various assembly techniques on this low-quality sample. We evaluated different approaches for de novo assemblies and discuss their value in relation to biological analyses. The high degree of variability between each de novo assembly method (assessed from the seven key metrics) highlights the importance of carefully devising the sequencing strategy to be able to carry out the desired analysis. Adding more data to genome assemblies does not always results in better assemblies so it is important to understand the nuances of genomic data integration explained here, in order to obtain cost-effective value-for-money when sequencing genomes.

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. Protocols for "Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal". protocols.io https://dx.doi.org/10.17504/protocols.io.bd3ri8m6 Copy   

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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: Hiseq 2000 Library Construction and Sequencing for RNA Seq

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. Hiseq 2000 Library Construction and Sequencing for RNA Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.38kgruw Copy   

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Authors: Yasmin Bar El
Group: GigaScience Press
Summary: Immunocytochemistry for morphology analysis. Staining of cell culture in order to obtain fluorescence images of the culture.

Proper citation: Yasmin Bar El 2018. Immunocytochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.tqdems6 Copy   

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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: This protocol provides an efficient DNA extraction and purification of ancient bones.

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. Extraction method B. protocols.io https://dx.doi.org/10.17504/protocols.io.iadcaa6 Copy   

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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: Southeast (SE) Asia is one of the most biodiverse regions in the world and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia’s genetic diversity is still poorly characterized. The growing interest in using environmental DNA (eDNA) to assess and monitor SE Asian species, in particular, threatened mammals - has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples.

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. Supporting data for "An expanded mammal mitogenome dataset from Southeast Asia". protocols.io https://dx.doi.org/10.17504/protocols.io.im6cc9e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bpyumpww Copy   

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Authors: Chang Li
Group: GigaScience Press, BGI
Summary: This protocol is used to clarity the process of total DNA extration for L. maculatus genome.

Proper citation: Chang Li 2018. DNA extraction for vertebrate tissues using Phenol:Chloroform:Isoamylol. protocols.io https://dx.doi.org/10.17504/protocols.io.ssyeefw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.sszeef6 Copy   

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Authors: Benjamin Istace, Anne Friedrich, Léo dAgata, 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
Summary: This protocol describes the library preparation for Illumina sequencing. It accompanies the 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 dAgata, 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. Illumina PCR-Free library preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.gvtbw6n Copy   

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Authors: Eric Carpenter
Group: GigaScience Press
Summary: Implemented by: Michael DeyholosThis protocol combines elements of standard CTAB, acid phenol, and silica-membrane protocols. The protocol was developed to extract total RNA from a wide range of plant species and tissues, and to do so in the smallest volume possible to yield >20 μg of total RNA using as little as 50 mg of fresh tissue. Maintaining a small extraction volume also allows for many samples to be processed in parallel in microcentrifuge tubes; at least 12 samples can be processed in 3–4 hours. The protocol has been used successfully with dozens of species, including tissues rich in polysaccharides and/or secondary metabolites. There are four organic extractions in total (three chloroform and one phenol:chloroform). For many species/tissues, the full set of chloroform extractions in the order specified is required to maximize RNA purity and to prevent phase inversion during the acid phenol extraction.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 8: CTAB/Acid Phenol/Silica Membrane Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4yfgxtn Copy   

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