Comparison of long and short 16S rRNA gene amplicon sequencing for agricultural soil and grain microbiomes using custom Kinnex libraries

Abstract Bacteria are an important component of environmental microbiomes, contributing to animal, plant, and soil health. The 16S rRNA gene is used as a barcode for taxonomic classification of bacteria and analysis of bacterial diversity in environmental samples. The 16S barcode region is ~1,500 bp and has nine hypervariable regions (V1-V9) that differ in their ability to distinguish various taxa. Short-read sequencing approaches enabled the targeting of subsets of these variable regions; however, long-read sequencing platforms can span all nine hypervariable regions, resulting in greater taxonomic resolution. We explored different DNA extraction procedures, DNA polymerases, and amplicon lengths to determine their effects on taxonomic resolution. We compared the full-length 16S barcode region (V1-V9) and subsets of the barcode (V3-V5 and V4-V5) using custom amplicons in the Pacific Biosciences (PacBio) Kinnex long-read sequencing platform, which has enhanced output over traditional PacBio amplicon sequencing. To capture differences in DNA quality and microbial complexity, two types of environmental DNA samples were assessed: 32 samples from agricultural soil and another 32 samples cultured from grain. The study demonstrates that custom amplicon sequencing using the PacBio Kinnex system can be implemented with relative ease, allowing for increased outputs over traditional custom amplicon sequencing. The greatest taxonomic resolution was achieved with longer amplicons; however, interactions between DNA quality, amplicon lengths, chimeric reads, community complexity, and taxonomic assignment/resolution should be considered. The samples themselves also represent unique microbiomes that are not well represented in the literature, adding to our knowledge of diverse microbes on grain and in soils.