title: Fairyland_2
design: DNA quality and integrity were comprehensively assessed prior to long-read library preparation. Fragment size distribution was evaluated using the Femto Pulse capillary electrophoresis system (Agilent Technologies), providing high-resolution sizing of DNA fragments (75 bp165 kb). Key metrics, including modal fragment size, DNA Integrity Number (DIN), and overall size distribution profiles, were generated to assess suitability for long-read sequencing. These parameters guided decisions on controlled fragmentation, with samples exhibiting modal fragment sizes >10 kb subjected to mechanical shearing to achieve optimal insert sizes for PacBio HiFi sequencing. SMRTbell sequencing libraries were prepared following the PacBio protocol Preparing 10 kb Libraries Using the SMRTbell Express Template Prep Kit 2.0 for Metagenomic Shotgun Sequencing (Pacific Biosciences), using 1.5 g of high-quality genomic DNA per library. Library preparation was conducted in accordance with manufacturer recommendations to ensure compatibility with PacBio HiFi sequencing chemistry.Briefly, input DNA fragments were subjected to end-repair to generate blunt-ended molecules with 5 phosphorylated termini, followed by A-tailing to add a single adenine residue to the 3 ends. These modifications facilitated efficient ligation of SMRTbell hairpin adapters. Following adapter ligation, exonuclease treatment was performed to selectively remove linear DNA molecules and incompletely ligated products, thereby enriching for intact circular SMRTbell templates. Sequencing primers complementary to the SMRTbell adapters were subsequently annealed to generate sequencing-ready libraries. Completed libraries underwent quality control prior to sequencing. Insert size distributions were verified using Femto Pulse capillary electrophoresis to confirm conformity with target fragment sizes. Library concentrations were determined using fluorometric quantification to ensure optimal loading of SMRT Cells. Adapter ligation efficiency was assessed to confirm successful library construction and suitability for sequencing.Libraries were sequenced individually without pooling to maximize read output per sample and to ensure sufficient sequencing depth for the detection of low-abundance microbial taxa.