Dataset DOI: 10.5061/dryad.73n5tb37z

Description of the data and file structure

This dataset contains raw Illumina sequencing reads (FASTQ format) derived from artificial pollen mixtures composed of three plant species: Eucalyptus baxteri, Echinacea arctotheca, and Prunus dulcis. The purpose of the experiment was to evaluate the accuracy of DNA-based taxonomic identification and quantification using targeted chloroplast hybrid capture followed by high-throughput sequencing.

The pollen was combined in varying proportions to create 14 artificial mixes, each with three replicates, resulting in 42 DNA libraries plus  negative controls. Each sample was assigned a unique internal barcode and dual index combination for multiplex sequencing.

Libraries were prepared using the NEBNext Ultra II kit with custom stubby Y-adapters and enriched using the OZBaits_CP V1.0 chloroplast bait set targeting 19 conserved chloroplast genes. The final library pool was sequenced on an Illumina HiSeq X Ten platform (2 × 150 bp reads).

Files and variables

File description:

Each sample is represented by two compressed FASTQ files: a forward read file (R1) and a reverse read file (R2), containing raw Illumina sequencing data. These files have already been demultiplexed based on the internal in-line barcodes used during library preparation.

The filenames follow a naming convention that includes:

• the mix ID (e.g., M07, M14) corresponding to a specific artificial pollen mixture, and
• the replicate ID (a, b, or c) indicating one of the three replicates per mix.

For example:
Poll_713_M7a_R1.fastq.gz and Poll_713_M7a_R2.fastq.gz represent the forward and reverse reads for replicate a of Mix M7.

Blank (negative control) samples follow a similar naming convention (e.g., Poll_760_EBC2_Blank_R1.fastq.gz).

Files:

File: Poll_716_M8a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_716_M8a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_715_M7c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_715_M7c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_713_M7a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_714_M7b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_713_M7a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_760_EBC2_Blank_R1_001.fastq.gz

File: Poll_760_EBC2_Blank_R2_001.fastq.gz

File: Poll_718_M8c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_714_M7b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_718_M8c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_722_M10a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_720_M9b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_723_M10b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_723_M10b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_721_M9c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_720_M9b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz 

File: Poll_721_M9c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_722_M10a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_724_M10c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_717_M8b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_730_M12c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_724_M10c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_719_M9a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_719_M9a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_717_M8b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_729_M12b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_728_M12a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_730_M12c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_729_M12b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_728_M12a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_738_M15b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_738_M15b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_739_M15c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_727_M11c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_726_M11b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_727_M11c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_739_M15c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_726_M11b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_737_M15a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_737_M15a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_741_M16b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_742_M16c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_741_M16b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_743_M17a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_742_M16c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_735_M14b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_743_M17a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_744_M17b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_735_M14b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_744_M17b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_745_M17c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_745_M17c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_740_M16a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_740_M16a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_725_M11a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_725_M11a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_746_M18a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_746_M18a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_747_M18b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_747_M18b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_750_M19b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_750_M19b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_748_M18c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_748_M18c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_753_M20b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_753_M20b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_752_M20a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_751_M19c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_752_M20a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_751_M19c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_731_M13a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_731_M13a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_734_M14a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_732_M13b_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_734_M14a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_732_M13b_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_754_M20c_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_733_M13c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_754_M20c_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_733_M13c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_749_M19a_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

File: Poll_749_M19a_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_736_M14c_Mix_E_baxteri_E_arctotheca_P_dulces_R2_001.fastq.gz

File: Poll_736_M14c_Mix_E_baxteri_E_arctotheca_P_dulces_R1_001.fastq.gz

Corresponding sample names in publication

Note that in the publication, the Mix ID was modified from M7-M20, to M1-M14.

Code/software

How to Open and Analyze the Data

The dataset consists of demultiplexed Illumina sequencing reads in compressed FASTQ format (.fastq.gz). These files can be opened in any text editor that supports gzip compression, but they are typically analyzed using command-line tools or within bioinformatics workflows.

A range of free and open-source software is available to view, quality-check, and analyze these files. Below is an overview of the tools used in this study, organized by processing step.

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1. Demultiplexing (already completed)

Software:

• bcl2fastq(Illumina): Used to demultiplex reads based on Illumina index sequences.
• Sabre: Used to demultiplex based on internal 8 bp barcodes.
  Note: Sabre was run allowing one mismatch, as all internal barcodes differed by at least two base pairs.

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2. Quality Filtering and Trimming

Software:

• Clumpify (BBTools suite): Used to remove PCR duplicates by grouping identical reads.
• AdapterRemoval: Used for trimming adapter sequences, filtering short/low-quality reads, and removing trailing A-tails. Reads shorter than 30 bp or with Phred scores <20 were removed.

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3. Quality Assessment

Software:

• FastQC: For checking per-base quality scores, GC content, and other quality metrics.
• MultiQC: For summarizing FastQC reports across all samples.
• seqtk, zcat, less: Command-line tools for quick inspection or subsampling of reads.

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4. Taxonomic Classification and Quantification

Software:

• Kraken2: Used for assigning taxonomic labels to reads via a k-mer-based approach.
• Bracken: Used for estimating taxon abundance from Kraken2 output.