title: Endobacteriome 16S rDNA Ambrosia artemisiifolia: leaf location 1
design: PCR amplification of the bacterial 16S rRNA gene (V3-V4 region) was performed using primers 322F (5-ACGGHCCARACTCCTACGGGA-3) and 796R (5-CTACCMGGGTATCTAATCCKG-3) (Chen et al., 2022). Reactions (25 L total volume) contained: template DNA (1 L, if needed diluted 1:10 to reduce PCR inhibition), ColorTaq polymerase (1.5 U; EurX), 1 EurX PCR buffer, 2 mM MgCl, 0.4 g/L BSA, 0.2 M each primer, and 0.2 mM dNTPs. Thermocyclic conditions: initial denaturation (95C, 3 min), 30 cycles of denaturation (95C, 30 s), annealing (51C, 30 s), and extension (72C, 30 s), with final extension (72C, 7 min). PCR products were verified by agarose gel electrophoresis (2% TBE gel). PCR products were purified using Agencourt AMPure XP magnetic beads and resuspended in Elution Buffer. Library construction, quality assessment (Agilent 2100 Bioanalyzer), and sequencing were performed by BGI Genomics using the DNBSEQ-G400 platform (paired-end, 300 bp reads; Tag Number: 30000). Per-sample sequencing yields ranged from 23.424.2 Mbp raw data, with >94% clean reads retained after quality filtering.