Qubit v2.0 fluorometer

Total RNA from each sample was isolated using a QiagenRNeasy Mini Kit according to manufacturer’s instructions. FastPrep-24 homogenizer (MP Biomedicals) was used to process approximately 30 μg of each sample (30 sec at 4.0 m/s). RNA quality and quantity was assessed using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA) and a Qubit v2.0 fluorometer (Life Technologies, Darmstadt, Germany), respectively.
For each sample, 250 ng of high-quality RNA (RIN value > 8) was used to construct nine barcoded sequencing libraries with the Illumina TruSeq RNA sample preparation kit (Low-Throughput protocol) according to the manufacturer’s instructions (Illumina, San Diego, USA). To increase the average library insert size, input RNA was chemical fragmented at 94 °C for 1 min.
The nine barcoded samples were equimolar-pooled and clustered template cDNA was sequenced in an Illumina HiSeq2000 platform with 209 cycles (101 cycles for each paired-read and seven cycles for the barcode sequences).

X. albilineans strain Xa-FJ1 was isolated from a leaf section originating from a diseased sugarcane plant of clone YG48 collected at Zhangzhou, Fujian Province, China [12 (link)]. A pure culture of this strain was grown with constant shaking at 200 rpm and 28 °C for 48 h in XAS liquid medium [19 (link)]. Bacterial genomic DNA was extracted from cultures of Xa-FJ1 using the SDS method [20 (link)]. Extracted genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified using the Qubit v.2.0 fluorometer (Life Technologies, Carlsbad, CA, USA).

Total RNA from each sample was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, USA) and small non-coding RNA molecules were retained by using 100% ethanol in the final washing steps. A FastPrep-24 homogenizer (MP Biomedicals) was used to process approximately 50 µg of each sample (30 sec at 4.0 m/s). A Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA) was used to check RNA quality, while its quantification was carried out with a Qubit v2.0 fluorometer (Life Technologies, Darmstadt, Germany). High-quality RNA samples (RIN value > 8.0) were used to construct RNA-Seq and miRNA-Seq sequencing libraries. For RNA-Seq, the Illumina TruSeq RNA sample preparation kit v2 (Illumina, San Diego, USA) was used to process 200 ng of total RNA in order to construct 48 sequencing libraries. Bar-coded libraries were paired-end sequenced (2 × 150 bp) in two lanes of an Illumina HiSeq2500 platform at the Tufts University genomic facility (TUCF Genomics, Boston, USA). For miRNA-Seq, the NEBNext Small RNA Library Prep Set (New England Biolabs, Beverly, USA) was used to construct 48 barcoded miRNA libraries from the same total RNA used for the RNA-Seq approach. Single-end sequencing (50 bp) was carried out in an Illumina HiSeq2500 at TUCF Genomics.

DNA was extracted from stool samples using a PowerSoil DNA isolation kit (Mo Bio Laboratories, Inc.) (4 (link)). Library preparation was done using a two-step PCR method to amplify the v4 region of the 16S rRNA gene (see Text S1 in the supplemental material). During the first step of PCR, primers PE16S_V4_U515_F (5′ ACACG ACGCT CTTCC GATCT YRYRG TGCCA GCMGC CGCGG TAA 3′) and PE16S_V4_E786_R (5′-CGGCA TTCCT GCTGA ACCGC TCTTC CGATC TGGAC TACHV GGGTW TCTAA T 3′) were used to target and amplify the v4 region, as well as to add second-step priming sites (40 (link), 41 (link)). Library size was confirmed at approximately 440 bp with a Qiaxcel Advanced system (Qiagen). Libraries were quantified with a Qubit v.2.0 fluorometer (Life Technologies, Inc.) and were pooled and denatured following the Illumina protocol. Paired-end sequencing (2 × 250 bp) was performed using MiSeq reagent kit V2 (Illumina) and a MiSeq sequencer (Illumina). All sequencing was done in a single run, with a Q score greater than Q30 for 93.1% of reads, and a cluster density of 856 ± 12 K mm⁻².

The intestinal content samples were thawed on ice, and then genomic DNA were separately extracted using the E.Z.N.A. Stool DNA kit (OMEGA, Bio-Tek, USA) based on the manufacturer's protocol and stored at −80°C. The integrity of the 13 DNA samples was assessed visually using agarose gel (containing ethidium bromide) electrophoresis on 1.0% and quantified using a Qubit v2.0 fluorometer (Life Technologies, Darmstadt, Germany). The DNA concentration was determined by using a fluorescence spectrophotometer (ES-2, Malcom, Japan).

The PCR results were validated in three adult zebra mussels from the Aragón Imperial Canal (Ebro River), and the PCR specificity was verified against several mollusc species found in overlapping distribution with zebra mussel: one individual of Spengler’s freshwater mussel (Margaritifera auricularia), two Asian clam (Corbicula fluminea) and one spike-topped apple snail (Pomacea sp.). Furthermore, two individuals of quagga mussel sampled in the Netherlands (52°42′N, 05°18′E) were also included. Whole bodies without shell were preserved in 70% ethanol until processed. DNA isolations were performed using the EZNA Mollusk DNA kit (Omega Bio-Tek), and DNA was eluted in a volume of 200 μl. DNA quality and quantity was verified by agarose gel electrophoresis, Qubit v2.0 fluorometer (Life technologies) and NANODROP spectrophotometer (Thermo Fischer Scientific).
In addition, to infer the possible presence of quagga mussel in waters of the Iberian Peninsula, we collected more than 4,000 dreissenid adult individuals in 2013. All individuals were collected in Ribarroja reservoir (Ebro River; first cited record of the zebra mussel in the Iberian Peninsula8 ; n = 3,013) and La Baells (Llobregat River; last cited record in the Iberian Peninsula23 ; n = 1,230) (see Supplementary Table S1).