16S V3-V4 Amplicon 2x300 Sequencing

2x300 paired-end sequencing data generated with both Cloudbreak and Cloudbreak Freestyle chemistries for low-diversity high-plexity 16S rRNA gene amplicons.


Libraries were prepared from ZymoBIOMICS Microbial Community Reference DNA (#D6305) and ZymoBIOMICS Fecal Reference with TruMatrix Technology™ (#D6323). DNA was extracted from the ZymoBIOMICS Fecal Reference sample using the ZymoBIOMICS DNA Miniprep kit (#D4300) with lysis tubes vortexed for 40 minutes.

Libraries were then prepared using a 2-step PCR workflow. V3-V4 regions of 16S rRNA genes were first amplified using 341F/805R primer pairs with either 3rd Party or Element Elevate specific PCR handles for 25 cycles, followed by amplification with IDT xGen UDI primer pairs for 3rd Party (#10005922) or Element Elevate (#10017037) for 6 cycles.

3rd Party libraries were sequenced as circular Adept with Cloudbreak chemistry for 2x300 cycles using High Ouput flowcells and enabling Low-Diversity High-Plexity mode to improve polony detection from low-diversity amplicon libraries. Linear Elevate libraries were sequenced with Cloudbreak Freestyle chemistry, for 2x300 cycles using High Output kits without the need for additional settings as diversity is built into the Index1 sequence in Elevate adapters. Reads were downsampled to 10,000-20,000 reads per sample.


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