Fig. 5: Comparison of library preparation protocols.

a β-diversity analysis – NMDS. The sample-wise Bray-Curtis distances between the samples were calculated and plotted using NMDS. Colors show the DNA isolation methods, while shapes indicate the library preparation protocols. b β-diversity analysis—PERMDISP. PCoA visualization of the dispersion values from the PERMIDISP results. The plot is faceted according to the DNA isolation methods and colors indicate the library preparation protocols. c β-diversity analysis—Heatmap. The sample-wise Bray-Curtis distances between the samples were calculated and visualized on a heatmap. The WGS data composition for the I and MN isolation kits is most similar to the bacterial composition from the Z V3-V4 library, followed by the ONT V1-V9 library. Notably, for both I and MN DNA isolation kits, the WGS and ONT V1-V9 libraries exhibit fairly similar compositional profiles. The Z DNA isolates produce relatively consistent microbial community profiles across different library preparation techniques. The PE V1-V3 library for Q DNA shows some similarity to WGS. The I and MN DNA samples exhibit significant overlap with the V1-V2 libraries prepared from I, MN, and Z DNA, with Z DNA showing a particularly high degree of overlap with itself. Additionally, Z DNA shows good overlap with the V1-V3 library compared to other libraries. The V3-V4 library consistently shows the least similarity to other libraries across all DNA isolation methods. The PacBio results are most similar to ONT, regardless of the DNA isolation kit used. When using Z DNA isolation method, PacBio shows significant similarity in microbial composition with other SRS amplicon-based sequencing results, particularly with V1-V2, V1-V3, and then V3-V4. WGS is significantly different. The Z-isolated DNA ONT V1-V9 libraries also show similar compositional similarities with these, with PacBio possibly showing a closer match. d Relative abundance of the top 20 phyla in each sample with. Rows indicate the DNA isolation methods, while columns indicate the library preparation protocols. e Barplot showing the top 20 phyla in the MCS samples, sequenced using Invitrogen DNA isolation kit, according to library preparation protocols, analyzed with minitax, DADA2, and Emu programs using several different databases.