Amplicon Sequencing of Forest Litter: Burnt Top Litter
1) Genomic DNA Quality Detection High-quality genomic DNA with good integrity is a prerequisite for library amplification and construction. The extracted genomic DNA was assessed using the following methods: a. Agarose Gel Electrophoresis for Genomic DNA Integrity: Electrophoresis bands were clearly visible with no significant degradation. b. Sample Pre-amplification PCR Detection (e.g., ITS2): Sample DNA templates, without the addition of spike-in reference DNA, underwent high-fidelity PCR pre-amplification targeting the sample detection region, with a standard fungal genomic DNA mix serving as a positive control. Amplification products were analyzed via agarose gel electrophoresis. Samples with qualified DNA quality control and successful PCR amplification were deemed acceptable. Amplification primers were designed according to the selected target region. For this project, the primer information for the target region (ITS2) is as follows: Primer F = Illumina adapter sequence 1 + GCATCGATGAAGAACGCAGC Primer R = Illumina adapter sequence 2 + TCCTCCGCTTATTGATATGC Where: Illumina adapter sequence 1 = AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG Illumina adapter sequence 2 = CTGTCTCTTATACACATCTCCGAGCCCACGAGACXXXXXXXXATCTCGTATGCCGTCTTCTGCTTG (XXXXXXXX represents the sample-specific index (barcode) sequence for sample identification. The index sequence corresponding to each sample is detailed in the fastq files.)  2) Estimation of Fungal ITS Copy Number Sample DNA and spike-in DNA with a known copy number at the lower limit of detection for the assay were used as PCR templates within the same amplification reaction mixture to amplify the target region. The fungal ITS copy number content in the PCR amplification products was estimated. Qualified samples were defined as having a fungal ITS copy number > 2E+03 copies/L of the original DNA solution.  3) Amplification of Target Region for Qualified Samples (e.g., ITS2) Sample DNA and spike-in reference DNA served as PCR templates and were added to the same amplification reaction mixture for target region amplification. An appropriate proportion of spike-in reference DNA was added to each sample, alongside positive control samples.  4) Addition of Sample-specific Index Sequences Sample-specific index (barcode) sequences were introduced to the library ends via high-fidelity PCR using primers containing the index sequences. This enables the pooling of multiple samples for downstream sequencing and allows subsequent bioinformatic analysis to distinguish samples based on their unique index sequences. Amplification products were checked by agarose gel electrophoresis.  5) Library Pooling and Purification Based on preliminary quantification results from agarose gel electrophoresis, libraries with their respective index tags were appropriately diluted. Precise library quantification was then performed using Qubit. Libraries were pooled in corresponding proportions (molar ratios) according to the sequencing depth requirements for different samples. The pooled library was purified and recovered via gel excision.  6) Library Quality Check The pooled library was analyzed on an ABI 3730 system to determine the insert size of the sequencing library, confirming it was within the 120-200 bp range without non-specific amplification, and to accurately quantify the final library concentration.