DOI: 10.5061/dryad.h18931zjq
title: The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species

description: DNA metabarcoding has become a powerful approach for analyzing complex
 communities from environmental samples, but there are still methodological
 challenges limiting its full potential. While conserved DNA markers, like
 16S and 18S, often are not able to discriminate among closely related
 species, other more variable markers – like the fungal ITS region, may
 include considerable intraspecific variation, which can lead to
 over-splitting of species during DNA metabarcoding analyses. Here we
 assess the effects of intraspecific sequence variation in DNA
 metabarcoding, by analyzing local populations of eleven fungal species. We
 investigated the allelic diversity of ITS2 haplotypes using both Sanger
 sequencing and high throughput sequencing (HTS) coupled with error
 correction with the software dada2. All focal species, except
 one, included some level of intraspecific variation in the ITS2 region.
 Overall, we observed a high correspondence between haplotypes generated by
 Sanger sequencing and HTS, with the exception of a few additional
 haplotypes detected using either approach. These extra haplotypes, often
 occurring in low frequencies, were likely due to PCR and sequencing errors
 or intragenomic variation in the rDNA region. The presence of
 intraspecific (and possibly intragenomic) variation in ITS2 suggest that
 haplotypes (or ASVs) should not be used as basic units in ITS-based fungal
 community analyses, but an extra clustering step is needed to reach
 species-level resolution.    
description: DNA was extracted from 11 fungal species, DNA metabarcoding using
 the gITS7 and ITS4 primers, amplicons normalised and paired-end
 sequenced on Illumina Miseq (2 *300 bp). The bio-informatics pipeline is
 provided in the files, together with the raw data, statististics codes and
 and the intermediate files needed to re-run all analyses. "The
 rawdata available in the directory
 ""Issakka-ITS_rawdata".
description: A Readme file ("Dryad_ReadMe_Issakka_ITSClustering") is
 provided and is accessible upon download of the data.
 Data submission accompanying the article "The influence of
 intraspecific sequence variation during DNA metabarcoding: A case study of
 eleven fungal species" The data package is
 structured 2 directories:  1. The raw sequencing data
  "Issakka-ITS_rawdata". 2 ITSClustering which
 contains 3 folders:  2.1 "Bioinformatics",
 contains the two map files "map_lib1.txt and "map_lib2.txt"
 for demultiplexing the two ITS MiSeq libraries (in Folder_name =
 "rawdata_metabarcodingITS2"), the file "batchfileDAD2"
 for running DADA2, the file "bioinformatics_ITS2Clustering"
 indicating the bioinformatics pathway for generating ASVs and the file
 "NamedHost_ASV.xlsx" is the OTU table, including the mock
 community and the 12 replicates. 2. 2
 "Sanger_sequences" contains both fasta and ABI formats of the
 forward and reverse raw Sanger sequences, together with the matching file
 (xls) allowing to link sequence ID to sample ID. 2.3
  "Final_alignments" contains the combined haplotypes from ASVs
 and Sanger sequencing. For each species a text file is provided. The zip
 folder "Haploype_network" contains individual files (nexus
 format) used to generate the network in PopArt.