Illumina MiSeq Sequencing was used for the 16S rRNA gene sequencing (≈200K reads per sample) by Omega Bioservices. Both the V1–V3 and the V3–V4 primer sets were used for PCR amplification by Omega Bioservices. The V1 and V2 regions have been historically used for identification of LAB [21 (link)]. All variable regions in the 16S rRNA gene have been reported to be effective in identifying bacterial communities in kefir through the 16S rRNA gene sequencing [22 (link)]. Library preparation type was KAPA HiFi PCR as per Omega Bioservices. Data was delivered via Illumina BaseSpace web site.
Basespace website
Manufactured by Illumina
BaseSpace® is a cloud-based genomics computing environment provided by Illumina. It serves as a platform for hosting, storing, and analyzing genomic data generated from Illumina sequencing instruments.
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4 protocols using basespace website
1
16S rRNA Gene Sequencing of Kefir Microbiome
2
MiSeq Library Preparation and Sequencing
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A pooled library (20 nM) and a PhiX control v3 (20 nM) (Illumina) were mixed with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 12 pM each. The resulting library was mixed with the PhiX control v3 (5%, v/v) (Illumina) and 600 uL loaded on a MiSeq® v2 (500 cycle) Reagent cartridge for sequencing. All sequencing procedures were monitored through the Illumina BaseSpace® website.
3
MiSeq Library Preparation and Sequencing
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A pooled library (20 nM) and a PhiX control v3 (20 nM) (Illumina) were mixed with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration of 12 pM’s each. The resulting library was mixed with the PhiX control v3 (5%, v/v) (Illumina) and 600 μL loaded on a MiSeq® v2 (500 cycle) Reagent cartridge for sequencing. All sequencing procedures were monitored through the Illumina BaseSpace® website.
4
Sequencing 16S rRNA Amplicon Library
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A pooled library prepared with the method described in the section 2.4.2 and a PhiX control v3 were mixed with HT1 buffer and 0.2N fresh NaOH to yield the final concentration at 6 pM each. The resulting library and PhiX control v3 was combined to give a 95% 16S rRNA gene amplicon library and 5% PhiX control. A MiSeq® v2 (500 cycle) Reagent cartridge (Illumina) was prepared according to the manufacturer’s instruction. Samples were sequenced using the Illumina MiSeq platform and sequencing procedures were monitored on the Illumina BaseSpace® website.
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