Analysis of Amplicon sequences of the RbcL chloroplast gene Locus

Annette Hynes and Adam Rivers
April 24, 2025
USDA-ARS-GBRU

Project outline

This project processed 4701 samples from 8 runs. The samples were multiplexed with combinatorial inline barcodes
and then second indexed with Illumina barcodes. The data were sequences in 2 x 151 mode on an Illumina Miseq

Data processing

The data were processed with bbtools, Ultraplex and Qiime2. The document stinkbug_rbcL_tutorial.md provides a general overview of the workflow and provides the code used.

Classifier training

The RbcL reference dataset used for Qiime2 classifier training is from Dubois et al (2022). The library is available in figshare.

Output files

• merged_repseqs_filtered.qzv--representative sequences generated from dada2.
• merged_table_filtered.qzv--ASV feature table.
• merged_taxonomy_output_filtered.qzv--taxonomy table.
• merged_taxa_bar_plots_filtered.qzv--bar plots of taxonomy.
• rbcl_biplot_taxonomy_filtered.qzv--emperor plot of Robust Aitchison's Principal Component Analysis (RPCA).

References

Dubois, B., Debode, F., Hautier, L. et al.(2022). A detailed workflow to develop QIIME2-formatted reference databases for taxonomic analysis of DNA metabarcoding data. BMC Genom Data 23:53 https://doi.org/10.1186/s12863-022-01067-5