Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
人、细菌和真菌扩增子的采集、处理并测序
发布: 2015年05月20日第5卷第10期 DOI: 10.21769/BioProtoc.1477 浏览次数: 13859
评审: Aksiniya AsenovaEmily CopeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
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Abstract
Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power for classifying common staphylococci in the skin. This protocol is adapted for 454 pyrosequencing of amplicons.
Keywords: Microbiome (微生物)
16S rRNA (16S rRNA)
ITS (智能交通系统)
Skin microbiome extraction (皮肤微生物提取)
Materials and Reagents
- Sample collection and DNA extraction
- Catch-All sample collection swabs (Epicentre, catalog number: QEC89100 )
- MasterPure yeast DNA purification kit (Epicentre, catalog number: MPY80200 )
- Yeast cell lysis buffer (Epicentre, catalog number: MPY80200)
- ReadyLyse (Epicentre, catalog number: R1810M )
- Promega DNA IQ Spin baskets (Promega Corporation, catalog number: U1221 )
- Stainless steel beads, 5mm (QIAGEN, catalog number: 69989 )
- MoBio PCR water (MoBio, catalog number: 17000-10 )
- 100% (200 proof) ethanol (Warner-Graham Company, catalog number: 64-17-5 )
- 70% ethanol made from 350 ml of 100% ethyl alcohol and 150 ml of MoBio water
- PureLink Genomic DNA Mini Kit (Life Technologies, InvitrogenTM, catalog number: K182002 )
- Catch-All sample collection swabs (Epicentre, catalog number: QEC89100 )
- PCR amplification
- Accuprime Taq polymerase HiFi (Life Technologies, InvitrogenTM, catalog number: 12346-086 )
- Forward and reverse primers with designated barcodes (IDT custom order)
- MinElute PCR purification kit (QIAGEN, catalog number: 28006 )
- QuantIT ds DNA assay, high sensitivity (Life Technologies, InvitrogenTM, catalog number: P7589 )
- Ampure (SPRI) beads, 60 ml kit (Agencourt, catalog number: A63881 )
- TE (pH 8.0) (Life Technologies, catalog number: AM9849 )
- Accuprime Taq polymerase HiFi (Life Technologies, InvitrogenTM, catalog number: 12346-086 )
Equipment
- 2.0 ml Safe-Lock Biopur individually sealed tubes (Eppendorf, catalog number: 0030 121.597 )
- Sterile scissors (VWR, catalog number: 82027-594 )
- Sterile tweezers (VWR, catalog number: 231-SA-SE )
- Safe-Lock PCR Biopur tube (Eppendorf, catalog number: 0030 123.344 )
- 15 and 50 ml conical tubes (Falcon, catalog numbers: 14-959-49D and 14-432-22 )
- 96-well thermocycler plates (USA Scientific, catalog number: 1402-9596 )
- Clear adhesive plate seals (USA Scientific, catalog number: 2978-2100 )
- Foil plate seals (USA Scientific, catalog number: 2923-0110 )
- Reagent reservoirs (USA Scientific, catalog number: 2320-2620 )
- Heated shaking block that holds Eppendorf tubes (Eppendorf Thermomixer C)
- Bead beater (Qiagen TissueLyser II, catalog number: 85300 )
- Bead beater adapters (Qiagen TissueLyser Adapter Sets, catalog number: 69982 )
- UV crosslinker (UVP, catalog number: CL-1000 )
- Microcentrifuge (Eppendorf, Centrifuge 5415D )
- Thermocycler (Applied Biosystems Veriti)
- Plate centrifuge (Beckman Coulter, Allegra 6KR)
- Fluorometer plate reader (Thermo Scientific, catalog number: 5210450 )
- Multi-channel pipettes (pre- and post-PCR designated)
- 96- well microplates for fluorescence-based assays (Life Technologies, catalog number: M33089 )
- Qubit 2.0 Fluorometer (Life Technologies, InvitrogenTM, catalog number: Q32866 )
- Qubit 2.0 Quantitation Starter Kit (Life Technologies, InvitrogenTM, catalog number: Q32871 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Oh, J. (2015). Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing. Bio-protocol 5(10): e1477. DOI: 10.21769/BioProtoc.1477.
Download Citation in RIS Format
分类
微生物学 > 微生物遗传学 > DNA > DNA 测序
微生物学 > 微生物-宿主相互作用 > 体内实验模型 > 哺乳动物
系统生物学 > 基因组学 > 测序
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