Miseq apparatus

Library preparations of the fragments, sequencing reactions, and preliminary analysis of the data were performed at the Center for Translational Genomics and Bioinformatics, Hospital San Raffaele, Milano, Italy. Briefly, for the preparation of the bar-coded libraries, TruSeq ChIP sample prep kit (Illumina) was used on the VH DNA samples isolated from cycles 1–4. A complementary scheme for bar-coding was implemented, in order to perform sequencing reactions from mixtures of subcycles 1 and 4 (run #1) and of subcycles 2 and 3 (run #2). The bar-coded samples were diluted to a final concentration of 10 pM and sequenced with 2 × 300 nt SBS kit v3 on an Illumina MiSeq apparatus.

The genotyping
of calli and
shoots of chicory lines with mutations in CYP71DD33 was performed by Sanger sequencing. A PCR product was amplified
from a small sample of the leaf tissue using the Phire Plant Direct
PCR Kit (Thermo Fisher) using primers targeting CYP71DD33. The PCR products were sent for Sanger sequencing and analyzed for
the presence of indels at the target sites. For detailed genotyping
of eight mutant lines showing large differences in the terpene profile,
PCR products were subsequently purified and cloned into the pJET1.2
vector (Thermo Fisher), and several cloned PCR products were sequenced
by Sanger sequencing. Sequence data were analyzed by SeqMan Pro software
(DNASTAR). Cas9 gene integration was checked by PCR
using specific primers that amplified an 800 bp fragment of the Cas9 gene.
To assess the efficiency of genome editing
for lines with mutations in CYP71DD35 and CYP71DD20, a PCR product was amplified from a small sample
of the leaf tissue using specific primers (Table S1). A nested PCR was done on each PCR product, and a final
third PCR was performed with barcoded Illumina primers to enable later
identification of the sequences. All of these PCR products were then
pooled and paired-end sequenced on an Illumina MiSeq apparatus. The
sequences were analyzed for the presence of indel mutations at the
target sites.

For multiplex PCR two customized Ion AmpliSeq primer pools for a total of 1255 amplicons were used (Life Technologies, Darmstadt, Germany). Primer sequences are given in Supplementary Table S5. The DNA input per reaction was 10 ng. Multiplex PCR was performed for 24 target genes (Table 2), most of which are involved in NF-κB signaling. The PCR program consisted of 99°C for 2 minutes, followed by 23 cycles of 99°C for 15 seconds and 60°C for 4 minutes. Libraries were prepared with the NEBNext Ultra DNA Library kit (NEB, Ipswich, UK) for sequencing on the Illumina platform. Purified libraries were quantified with the KAPA library quantification kit (Peqlab, Erlangen, Germany). Library size and quality was determined with the Agilent Bioanalyzer high sensitivity DNA assay (Agilent, Santa Clara, USA). Sequencing was performed on an Illumina MiSeq apparatus with 150 bp paired end reads.

Extracted DNA was used as input for PCR amplification of the ITS region of the rDNA. The Illumina ITS Primer Constructs (ITS1f-ITS2) for the amplification were fused with Golay indices and adapter sequences as described in the Earth Microbiome Project protocol (Thompson et al., 2017 (link))¹. The PCR (94°C, 3 min/35 × 94°C, 30 s; 52°C, 30 s; 72°C, 60 s/72°C, 10 min) was performed on a S1000™ Thermal Cycler (BIORAD) in 50 μl reactions using the Platinum™ PCR SuperMix (Thermo Fisher Scientific). After purification using NucleoMag NGS Clean-up and Size Select (Macherey-Nagel), size distributions and concentrations of the PCR products were analyzed on a TapeStation 2200 (Agilent Technologies). Final libraries were pooled in equimolar amounts and prepared for Illumina Sequencing using the MiSeq Reagent Kit v2 (Illumina) following manufacturers’ instructions. Run plan and reagents were adapted according to Caporaso et al. (2012) (link). Sequencing was performed on a MiSeq apparatus (Illumina) with 251 cycles.

Genomic DNA was isolated by the CTAB method (Wilson 2001 ). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer instructions. The genome of Acidovorax sp. A1169 was sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1 μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010 ).

Sequencing libraries were prepared from purified phage DNA using the Nextera XT DNA Library Preparation Kit and sequenced on an Illumina MiSeq apparatus. The resulting sequencing reads were de novo assembled by the pipeline A5-miseq version 20150522⁵² to obtain an initial sequencing depth. Given their small size and the high-throughput capabilities of the Illumina platform, viral genomes are usually sequenced with very high depth, causing an unnecessary complexity for subsequent de novo assembly^{53 (link)}. Consequently, the reads of each sequenced genome were randomly down-sampled by seqtk (https://github.com/lh3/seqtk) to obtain assemblies (also with A5-miseq version 20150522) having around 100X of sequencing depth.
We compared the resequenced phage genomes 44RR2.8t, 31, 56 and 65 by generating kmers with a length of 300 nt and then mapping them with BWA version 0.7.12-1039^{54 (link)} to reference sequences. Mutations were called by using samtools version 0.1.19-44428cd^{55 (link)} and VarScan version 2.4.2^{56 (link)}. Finally, the effect of mutations was evaluated based on SnpEff version 4.2^{57 (link)} (with the reference sequences added beforehand in the database).