Illumina NovaSeq X Plus sequencing

Stages

1. 1.
   Cold treatment and time-course sampling(functional_characterization)

   This stage establishes the perturbation and sampling framework needed to measure how cold stress responses change over time.

   Selection: Expose oil palm seedlings to cold and collect fresh leaf samples across exposure durations for downstream physiological and genetic analysis.

2. 2.
   Physiological parameter measurement(secondary_characterization)

   This stage captures phenotypic consequences of cold stress that can be compared with gene-expression changes.

   Selection: Measure antioxidant enzyme activity, ROS levels, photosynthetic pigment ratios, and photosynthetic efficiency under cold exposure.

3. 3.
   RNA sequencing(functional_characterization)

   This stage generates transcriptomic data for differential expression and pathway analysis.

   Selection: Sequence leaf-sample transcriptomes using the Illumina NovaSeq X Plus platform.

4. 4.
   Read quality filtering(decision_gate)

   This stage filters raw reads before downstream analysis to improve data quality.

   Selection: Remove adapter-containing reads, reads with >10% unidentified nucleotides, and reads where >50% of bases had Phred scores ≤20.

5. 5.
   Reference-guided transcriptomic interpretation(secondary_characterization)

   This stage converts filtered sequencing data into interpretable gene and pathway changes associated with cold stress.

   Selection: Analyze expression against reference genome GCF_000442705.2 and identify DEGs and enriched pathways.

Steps

1. 1.
   Subject oil palm seedlings to cold treatment

   Induce cold stress for downstream physiological and transcriptomic analysis.

   The perturbation must occur before any response measurements can be collected.

2. 2.
   Collect fresh leaf samples across exposure durations

   Capture time-resolved material for physiological and gene-expression analysis.

   Sampling follows treatment so that duration-dependent responses can be measured.

3. 3.
   Measure physiological stress-response parameters

   Quantify antioxidant, ROS-related, and photosynthetic responses to cold stress.

   These measurements characterize phenotypic stress responses from the collected samples before integrating them with transcriptomic results.

4. 4.
   Sequence samples on Illumina NovaSeq X Plussequencing platform

   Generate RNA-seq reads for transcriptomic analysis.

   Sequencing is required before computational read filtering and expression analysis.

5. 5.
   Filter raw reads with fastpread preprocessing software

   Remove adapter-containing and low-quality reads before downstream transcriptomic analysis.

   Quality filtering follows sequencing because raw reads must be cleaned before reliable DEG and pathway analysis.

6. 6.
   Analyze filtered reads against reference genome and identify DEGs and enriched pathways

   Convert filtered sequencing data into differential-expression and pathway-level interpretations of cold response.

   This analysis depends on having filtered reads and a specified reference genome.