Illumina HiSeq 2000 sequencing; qiita_ptid_104:990.KA2F.C.04 - SRA

ERX1630072: Illumina HiSeq 2000 sequencing; qiita_ptid_104:990.KA2F.C.04
1 ILLUMINA (Illumina HiSeq 2000) run: 106,528 spots, 15.8M bases, 10.4Mb downloads

Design: This analysis was done as in Caporaso et al 2011 Genome research. The PCR primers (F515/R806) were developed against the V4 region of the 16S rRNA (both bacteria and archaea), which we determined would yield optimal community clustering with reads of this length using a procedure similar to that of ref. 15. [For reference, this primer pair ampli_es the region 533_786 in the Escherichia coli strain 83972 sequence (greengenes accession no. prokMSA_id:470367).] The reverse PCR primer is barcoded with a 12-base errorcorrecting Golay code to facilitate multiplexing of up to _1,500 samples per lane, and both PCR primers contain sequencer adapter regions. The three sequencing primers include two for reading in from each end of the amplicon and a third for reading the barcode. Because of technical limitations at the sequencing facility, only part of the barcode was sequenced, so we were unable to exploit the error-correcting properties fully; however, even with partial barcodes we were able to resolve the samples, demonstrating the robustness of the approach. It is important to note that this primer collection allows for sequencing of paired-end reads, but the downstream data analyses are not yet capable of supporting paired-end reads. Our results illustrate interesting and correlated patterns based on analysis of the unpaired reads (i.e., _ and _ diversity evaluations based on the 5_ only and 3_ only reads independently achieve similar results, suggesting that 100 bases in this region of the 16S gene can allow for successful screening and comparison of microbial communities). The reads generated from these PCR primers are both identi_ed as _recommendedî regions by Liu et al. (15). Polymerase Chain Reaction. Sample preparation was performed similarly to that described by Costello et al. (1). Brie_y, each sample was ampli_ed in triplicate, combined, and cleaned using the MO BIO 96 htp PCR clean up kit. PCR reactions contained 13 _L MO BIO PCR water, 10 _L 5 Prime Hot Master Mix, 0.5 _L each of the forward and reverse primers (10 _M _nal concentration), and 1.0 _L genomic DNA. Reactions were held at 94 C for 3 min to denature the DNA, with ampli_cation proceeding for 35 cycles at 94 _C for 45 s, 50 _C for 60 s, and 72 _C for 90 s; a _nal extension of 10 min at 72 _C was added to ensure complete ampli_cation. Cleaned amplicons were quanti_ed using Picogreen dsDNA reagent in 10 mM Tris buffer (pH 8.0). A composite sample for sequencing was created by combining equimolar ratios of amplicons from the individual samples, followed by gel puri_cation and ethanol precipitation to remove any remaining contaminants and PCR artifacts.

Submitted by: University of California San Diego Microbiome Initiative (University of California San Diego Microbiome Init)

Study: Spatial scale drives patterns in soil bacterial diversity.

PRJEB15061 • ERP016752 • All experiments • All runs

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Most soil processes, including microbial diversity and CO2 efflux, are extremely variable across spatial scales. The destructive and labor-intensive nature of traditional soil sampling and processing methods have hampered broad investigation of spatial heterogeneity in soil function. We collected microsamples of soil (<3 g each) that provided enough material for DNA extraction to characterize the microbial community while reducing sample processing time that can result in DNA degradation and will dramatically enhance the spatial resolution of the of the diversity metrics. We collected twenty-five soil samples (0.7 cm dia X 5 cm deep) in a 10x10 cm grid at five points along a 36 m transect in each of six 36 x 20 m plots representing a low-diversity perennial grassland dominated by Panicum virgatum (fertilized and unfertilized treatments). The aim is to correlate microbial community structure with abiotic factors, which will help build a mechanistic understanding of feedbacks between soil microbes and ecosystem C fluxes.

Sample: Fermilab_spatial_study; 990.KA2F.C.04

SAMEA4369571 • ERS1281020 • All experiments • All runs

Organism: soil metagenome

Library:

Name: 990.KA2F.C.04

Instrument: Illumina HiSeq 2000

Strategy: OTHER

Source: METAGENOMIC

Selection: PCR

Layout: SINGLE

Construction protocol: EMP V4 515f,806rbc protocol

Experiment attributes: (show all 17 attributes...) (hide...)

barcode: ATAGAGGCCATT

center_name: ANL

center_project_name: Fermilab_spatial_study

emp_status: EMP

illumina_technology: HiSeq

linker: GT

pcr_primers: FWD:GTGCCAGCMGCCGCGGTAA; REV:GGACTACHVGGGTWTCTAAT

primer: GTGTGCCAGCMGCCGCGGTAA

run_center: ANL

run_date: 2012

run_prefix: lane3_NoIndex_L003_sequences

samp_size: .1,g

sample_center: ANL

sequencing_meth: sequencing by synthesis

study_center: CCME

target_gene: 16S rRNA

target_subfragment: V4

Runs: 1 run, 106,528 spots, 15.8M bases, 10.4Mb

Run	# of Spots	# of Bases	Size	Published
ERR1559316	106,528	15.8M	10.4Mb	2016-08-15

ID:: 2920566

SRA

Sequence Read Archive

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