The hypervariable V4 region of the small subunit rRNA gene was the target of amplification using the PCR primer pair 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR primers had incorporated an Illumina flow cell adapter at their 5′ end and the reverse primers contained 12 bp unique ‘barcode’ for simultaneous sequencing of several samples62 (link). PCR, including No-Template Controls (NTCs) for each barcoded primer, was performed as previously reported with the exception of the BSA at 10 mg/ml concentration per reaction23 (link). Only samples whose NTCs yielded an undetectable PCR amplification were retained for further analysis.
Flow cell adapter
Manufactured by Illumina
Sourced in United States
The flow cell adapter is a device designed to securely hold and position a flow cell within a sequencing instrument. Its primary function is to provide a stable and precise interface between the flow cell and the instrument components, ensuring accurate sample handling and data collection during the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Picogreen, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
16s metagenomics workflow, by Illumina (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Agencourt ampure xp kit, by Beckman Coulter (1 mentions)
Miseq lane, by Illumina (1 mentions)
8 protocols using flow cell adapter
1
16S rRNA Gene Amplification and Sequencing
2
Analysis of Gut Microbiome Composition via 16S rRNA Sequencing
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On day 50, feces were collected in a sterile tube filled with 1 mL PBS and then immediately frozen at −80 °C. For isolation of DNA, 100–300 mg of fecal material was ground with silica beads and extracted with a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. PCR products for the V4 region of the 16S rRNA gene were amplified with region-specific primers that included the Illumina flowcell adapter (Illumina, San Diego, CA, USA) sequences and 12-base barcodes on the reverse primer. PCR production of the bacterial DNA template was quantified using Invitrogen’s PicoGreen. Taxonomic classification of 16S rRNA targeting amplicon reads and the eight most abundant bacterial sequences was performed using Illumina 16S Metagenomics workflow in the Miseq Reporter software curated by the GreenGene taxonomic database (https://basespace.illumina.com/analyses/ ). Alpha diversity was calculated based on the Shannon index for richness and evenness of bacterial sequences at rarefraction depth reads of the operational taxonomic unit sample.
3
Comprehensive Microbial Community Profiling
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PCR amplifications employed primer sets (attached with Illumina flow cell adapter) that targeted the 16S rRNA gene of bacteria and archaea, an internal transcribed spacer (ITS) region of fungi, and the 18S rRNA gene of other eukaryotes (Table 2 ). PCR amplification was carried out in a total volume of 20 µL containing 40 ng (10 ng/µL) microbial template genomic DNA, 0.6 µL (10 µM) each of forward and reverse primers, 4.8 µL PCR-grade water and 10 µL 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Boston, MA, USA). PCR conditions were as follows: 95 °C for 5 min (initial denaturing step), 30 cycles of 20 s at 98 °C, 20 s at 58 °C, and 30 s at 72 °C, followed by a final extension step at 72 °C for 5 min. Amplicons were quality-tested and size-selected using gel electrophoresis (1.2% (w/v) agarose concentration and 1× TAE run buffer). All PCR was conducted after pooling triplicate samples of total DNA isolates. PCR products were cleaned-up using AMPure XP beads (Agencourt® AMPure® XP kit; Beckman Coulter, Brea, CA, USA) according to the Illumina MiSeq protocol for amplicon preparation. The following steps of library preparation and sequencing were performed by the DNA sequencing section of the Okinawa Institute of Science and Technology (OIST) Graduate University. Sequencing was done on an Illumina MiSeq using MiSeq Reagent Kit V3.
4
Soil Microbiome Amplicon Sequencing
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The hypervariable V4 region of the small subunit rRNA gene was the target of amplification using the PCR primer pair 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR primers had incorporated an Illumina flow cell adapter at their 5' end and the reverse primers contained 12bp unique 'barcode' for simultaneous sequencing of several samples 63 . PCR, including No-Template Controls (NTCs) for each barcoded primer, was performed as previously reported with the exception of the BSA at 10mg/ml concentration per reaction 25 (link) . Only samples whose NTCs yielded an undetectable PCR amplification were retained for further analysis.
5
STARR-seq Library Amplification and Sequencing
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We amplified 5 ng of the human STARR-seq (HSS) library with the following primers: P5-STARR-AG-ass-f and P7-STARR-ass-r (Supplemental Table 8 ). These primers add a sample-specific barcode and Illumina flow cell adapters. We then spiked the library into a NextSeq Mid 300 cycle kit with paired-end 149 bp reads and a 20 bp index read (which captured the 15 bp barcode as well as 5 bp of extra sequence to help filter for read quality), using the following custom primers: Read1 as STARR-AG-seq-R1, Read2 as spacer-seq-R2, Index1 as pLSmp-ass-seq-ind1 and Index2 as STARR-AG-ind2 (Supplemental Table 8 ).
6
Two-Step PCR Amplification of V4 Region
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All extracted DNA samples were subjected to a two-step PCR amplification with PCRBIO Hot Start VeriFi master mix. In the first thermal cycling program, the V4 region was amplified using 515f and 806r primers [26 (link)] appended to sequences CS1 and CS2, respectively using the same program as for the fungi. In the second program, barcodes and Illumina flowcell adapters were appended to the PCR1 amplicons with the same program used for the fungal libraries.
7
STARR-seq Library Amplification and Sequencing
8
Soil Metabarcoding of Cyst Nematode Communities
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Surface-sterilized cysts were crushed in a small centrifuge tube using a pestle. DNA was isolated from the macerated cysts according to a modified CTAB protocol described in Hu et al. (2017) (link). The DNA extracted from the 50 pooled cysts collected from each plot was used to construct a single metabarcode library, with four replicates per crop sequence at each collection time point. The PCR amplification, library preparation, and sequencing were conducted at the University of Minnesota Genomic Center, Saint Paul, MN, United States (Gohl et al., 2016 (link)). The universal bacterial primers targeting the 16S rRNA V4 region with primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) were used. The Illumina index and flow cell adapters were amplified together with the V4 primers during the first step PCR, and dual-index barcode sequence was added at the second PCR step (Gohl et al., 2016 (link)). The samples from the same year were pooled and sequenced on one Illumina MiSeq lane with the 2 × 300 bp kit. For each paired-end lane of MiSeq, additional samples were included in order to assess the PCR and sequencing error in the downstream pipeline, including blank control samples, a mock community sample, which had equal amount of DNA isolated from pure bacterial cultures, and nine randomly chosen cyst samples for technical replicates.
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