Library preparation and sequencing
For sequencing, we amplified a
short hypervariable fragment of the 18S SSU rRNA V7 region
(~100 – 110 bp) with 18S_allshorts primers (Forward
5’-TTTGTCTGSTTAATTSCG-3’, and Reverse 5’-GCAATAACAGGTCTGTG-3’)
(Guardiola et al., 2015). Broad coverage within Eukarya was verified
with the Arb Silva TestPrime function
(Klindworth et al., 2013). To
enable pooling of samples after amplification, primers were pre-tagged
with an 8-base oligonucleotide, wherein at least 3 nucleotides differed
between tags. The primers also contained a leading 5’-end variable
number of degenerate nucleotides (N, 2 ≤ 4) to increase sequence
variability and hence Illumina sequencing quality (Wangensteen et al.,
2018). PCR amplification and all downstream processing from here and
onwards were conducted twice. First as a pilot with a limited number of
samples and negatives (N1 = 79), and
finally with a full set of samples and negatives (N2 =
456). Samples were processed using the exact same protocol unless stated
otherwise.
PCR amplifications were performed
in 20 µL reactions with 10.00 µL AmpliTaq Gold ™ Master Mix (Applied
Biosystems), 0.16 µL Bovine Serum Albumin (BSA, 20 µg
µL-1), 5.84 µL ultrapure MQ water, 2.00 µL of
18S_allshorts Forward and Reverse primer mix (2.5 µM each), and 2.00 µL
DNA template. Thermal cycling consisted of an initial denaturation step
(10 min, 95°C), and 35 cycles of denaturation (30 s, 95°C), annealing
(30 s, 45°C) and elongation (30 s, 72°C). For each PCR plate, a
subsample of real samples (n = 9) and a PCR negative were tested on a
1% agarose gel to verify amplification and a lack thereof,
respectively. PCR amplicons were then pooled, purified with MinElute ™
spin-columns (Qiagen, Hilden, Germany) and quantified using the
broad-range dsDNA assay on a Qubit 4™ fluorometer (Invitrogen by
ThermoFisher). Sequencing-ready libraries were prepared from purified
pools in accordance with the NEXTflex™ PCR-Free DNA Sequencing Kit (Bioo
Scientific, Austin, Texas, USA). Here, DNA templates (3000 ng total for
each library) were first purified by size (retaining fragments
>150 bp) with magnetic Agencourt AMPure XP beads (Beckman
Coulter Genomics, California, USA), then adenylated, and ligated with
Illumina-compatible adapters. We used several Illumina-compatible
adapters (NEXTflex™ DNA Barcode Adapter, Bioo Scientific) to distinguish
libraries of 96 samples. The libraries were quantified by qPCR using the
NEBNext® Library Quant Kit for Illumina® (New England Biolabs,
Massachusetts, USA) to verify successful preparation. For the pilot
sequencing one library of 79 samples (75 real and 4 extraction
negatives), were sequenced using 150 Paired-End (PE) chemistry on a
HiSeq 4000 platform (Novogene Co., Ltd.). For the full sequencing run,
five libraries with a total of 456 samples (437 real and 19 extraction
negatives), were sequenced using 150 PE chemistry on two lanes of a
NovaSeq6000 platform (Novogene Co., Ltd.).