DOI: 10.5281/zenodo.17304914
title: Metabarcoding plant mock community dataset : Towards quantitative DNA Metabarcoding: A method to overcome PCR amplification bias

description: This dataset comprises DNA metabarcoding sequences obtained from three mock communities of alpine plants. These communities were designed to investigate quantification and amplification biases in such experiments. The Sper01 primer pair (Taberlet et al., 2007; Taberlet et al., 2018) was used to target the chloroplast trnL (P6 loop) marker. Each mock community was sequenced as a separate Illumina library, resulting in three independent libraries corresponding to three community compositions. High-throughput sequencing was carried out by Fasteris SA (Plan-les-Ouates, Switzerland) on an Illumina NextSeq platform using the Metafast library preparation protocol.

The dataset includes sequences from 13 alpine Spermatophyta species.

 




Species (Full name)
Short form
Amplicon length (bp)
GC content (%)
Total DNA concentration (ng/µl)
Rank (MG)




Briza media
Bme
53
39.6
183.00
1


Rosa canina
Rca
51
31.4
50.80
2


Lotus corniculatus
Lco
55
38.2
65.20
3


Populus tremula
Ptr
68
25.0
31.40
4


Salvia pratensis
Spr
46
26.1
24.40
5


Lonicera xylosteum
Lxy
46
32.6
45.80
6


Fraxinus excelsior
Fex
39
33.3
22.40
7


Acer campestre
Aca
56
39.3
12.20
8


Capsella bursa-pastoris
Cbp
48
45.8
38.80
9


Geranium robertianum
Gro
53
34.0
15.00
10


Carpinus betulus
Cbe
61
27.9
9.14
11


Abies alba
Aal
47
44.7
3.58
12


Rhododendron ferrugineum
Rfe
46
30.4
3.90
13




Table 1. Plant species included in the three mock communities and their characteristics for the Sper01 marker.Total DNA concentrations were measured by Qubit after extraction. “Rank (MG)” corresponds to decreasing abundance in the MG (geometric) community.

Leaf samples were collected in 2021 from the Chartreuse and Belledonne massifs (French Alps), dried in silica gel, and DNA was extracted using the CTAB protocol (Doyle, 1990), except for Carpinus betulus, which was extracted using the DNeasy Plant Mini Kit (Qiagen).

Absolute quantification of the Sper01 chloroplast target DNA was obtained using droplet digital PCR (ddPCR; Bio-Rad QX200 system) to account for interspecific variation in chloroplast copy number. For each species, the number of target copies per nanogram of total DNA was estimated. Based on these quantifications, three mock communities were prepared:





MU (Uniform community) – equal concentration of target DNA for each species;



Forward sequence file: 220930_NB501850_A_L1-4_GWM-1750_R1.fastq.gz

Reverse sequence file: 220930_NB501850_A_L1-4_GWM-1750_R2.fastq.gz

Demultiplexing metadata file: GWM-1750_U1.ngsfilter





MT (Total-DNA community) – equal concentration of total genomic DNA for each species;



Forward sequence file: 220930_NB501850_A_L1-4_GWM-1746_R1.fastq.gz

Reverse sequence file: 220930_NB501850_A_L1-4_GWM-1746_R2.fastq.gz

Demultiplexing metadata file: GWM-1750_U1.ngsfilter





MG (Geometric community) – target DNA concentrations following a geometric sequence (1, 1/2, 1/4, ...).



Forward sequence file: 220930_NB501850_A_L1-4_GWM-1750_R1.fastq.gz

Reverse sequence file: 220930_NB501850_A_L1-4_GWM-1750_R2.fastq.gz

Demultiplexing metadata file: GWM-1750_U1.ngsfilter




Each community was amplified in 4x20 PCR replicates. Each series of 20 is spiked with a different amount of the following synthetic sequence:

accctcagcctcgcccaaggttgaattatgaaacctgtgacggtcgggtc