Dataset: Amplicon sequencing of ammonia oxidizing archaea amoA gene

Name: Amplicon sequencing of ammonia oxidizing archaea amoA gene from R/V Kilo Moana KM1314 in the North Pacific Ocean, Aug-Sept 2013 (Nitrification and Planktonic Biodiversity project)
Published: 2017-01-04
Keywords: oceans

Cite This Dataset

Spatial Extent: N:50 E:-126 S:22.75 W:-158.5

Project:

Significance of nitrification in shaping planktonic biodiversity in the ocean (Nitrification and Marine Planktonic Biodiversity)

Program:

Dimensions of Biodiversity (Dimensions of Biodiversity)

Principal Investigator:

Dr David A. Stahl (University of Washington, UW)

Co-Principal Investigator:

E. Virginia Armbrust (University of Washington, UW)

Allan Devol (University of Washington, UW)

Anitra E. Ingalls (University of Washington, UW)

James W. Moffett (University of Southern California, USC)

BCO-DMO Data Manager:

Nancy Copley (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version Date:

2017-01-04

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Amplicon sequencing of ammonia oxidizing archaea amoA gene from R/V Kilo Moana KM1314 in the North Pacific Ocean, Aug-Sept 2013 (Nitrification and Planktonic Biodiversity project)

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Description

Amplicon sequencing of ammonia oxidizing archaea amoA gene data from R/V Kilo Moana KM1314 in the North Pacific Ocean (Seattle to Honolulu, including Line P, Station ALOHA).

Methods & Sampling

We determined the community diversity for nitrite max samples at stations 1, 3, 5, 10, 11, 13, 14, 15, and 16 and chlorophyll max samples at station 8 (55 m). Marine AOA amoA genes were amplified from template DNA (~20 ng/uL) using a two-step enrichment procedure for target amplification and sample barcoding on a Fluidigm Access Array (Fluidigm Inc., San Francisco CA). Gene targets were amplified using primer pairs ArchAmoA_1F and ArchAmoA1R and sample specific Golay primers (Francis et al. 2005). Amplified products were analyzed by gel electrophoresis to ensure the correct amplicon size, quantified using pico-green, pooled to an equal molar concentration and sequenced in pair-end 300 bp mode using the Illumina HiSeq Platform. Resulting sequences were assessed for quality using fastq_eestats and trimmed at the 5’ (5bp) and 3’ (225bp) ends (Edgar 2010). Trimmed reads were then quality filtered in QIIME using the recommendations described by Bokulich et al. (2013). The resulting reads were then analyzed using the UPARSE pipeline with an OTU clustering identity of 95% (Edgar 2013). Representative OTU sequences were analyzed against a reference database of AOA amoA genes in the ARB software using the parsimony tool for insertion (Ludwig et al. 2004; Pester et al. 2012). Sample-OTU matrices from UPARSE were converted to biom format and rarified to 1000 reads per sample using the single_rarfy.py script in QIIME. The resulting output file was then used for calculation of Bray-Curtis similarity values between samples and hierarchical clustering using the package vegan in R.

Data Processing Description

BCO-DMO Data Processing Notes:

- added conventional header with dataset name, PI name, version date, reference information
- renamed parameters to BCO-DMO standard
- converted lat and lon to decimal degrees

Data Files

File
amoA_reads.csv (Comma Separated Values (.csv), 735 bytes) MD5:372d3a6796bbaf838920317c18eeaadc Primary data file for dataset ID 672336

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1046017

Deployments

Deployment	Synonyms	Start Date	Platform	Investigator
KM1314		2013-08-07	R/V Kilo Moana	Anitra E. Ingalls (Chief Scientist)	data info

Instruments

Automated DNA Sequencer

Supplied Name: Illumina HiSeq Platform

Supplied Description:

Instrument Type

Generic Name: Automated DNA Sequencer

Acronym: Automated Sequencer

Generic Description:

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

Thermal Cycler

Supplied Name:

Supplied Description:

Instrument Type

Generic Name: Thermal Cycler

Generic Description:

A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.

(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
cruise_id	cruise identifier	unitless	cruise_id
sta	station number	unitless	sta
depth	sampling depth	meters	depth
lat	latitude; north is positive	decimal degrees	lat
lon	longitude; east is positive	decimal degrees	lon
Arch_OTU95_1_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OTU (operational taxonomic unit) 1	amplicon reads	no_bcodmo_term
Arch_OTU95_2_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OTU 2	amplicon reads	no_bcodmo_term
Arch_OTU95_5_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OTU 5	amplicon reads	no_bcodmo_term
Arch_OTU95_29_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OTU 29	amplicon reads	no_bcodmo_term
Arch_OTU95_34_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OTU 34	amplicon reads	no_bcodmo_term
Arch_OTU95_38_reads	amplicon sequencing reads of AOA amoA gene (encoding alpha unit of ammonia monooxygenase) for OUT 38	amplicon reads	no_bcodmo_term

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Database

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Dataset: Amplicon sequencing of ammonia oxidizing archaea amoA gene