Earray platform

A customized whole genome microarray was designed using the Agilent earray platform (https://earray.chem.agilent.com/earray/). Three probes for each gene were designed to investigate expression of the whole genome (3960 genes) of P. inhibens DSM 17395 and the derived mutants, of 26 internal controls (mainly putative housekeeping genes) and 536 Agilent controls, using the Agilent Technologies Design Wizard for a slide with eight arrays (8 × 15 K). Chosen probe features were length of 60 bp, Antisense, 3′ bias, probes layout randomized and best distribution.

The Nb-105k microarray design is registered in NCBI GEO repository under Platform accession number GPL21307 and is also publically available for ordering from Agilent eArray platform under ID 066813. In this design, each microarray probe is annotated with the original ID of the appropriate contigs from transcriptome v.3 or transcriptome v.5. Whenever the reverse complement of the original sequence was used for probe design (according to the results of sense strand identification performed in the current study), the contig ID has an RC_prefix. To download the original Agilent design files, with probe sequences or to order the Nb-105k microarray, go to https://earray.chem.agilent.com/earray/ then click the “Published Designs” link button on the right and select N. benthamiana from the species list.
The final microarray probes were also mapped to transcriptomes v.3 and v.5 to provide the links between two transcriptomes datasets. For this, each probe sequence was used as a query in a megablast similarity search against a local database created for a transcriptome v.3 or v.5, transcriptome, respectively, with the following parameters: e-value <0.0001, word size = 7, 65 % identity, no masking for low complexity sequences. Best hit for each search was reported in Additional file 2: Table S1 in addition to the primary targets (those for which the probes were designed).

The bottlenose dolphin microarray used in this work is a custom 44K oligonucleotide array designed using Agilent’s E-array platform (Amidad #028889) [13 (link)]. Details on the array are available in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (Platform GPL17696). The array included 24,418 unigene sequences obtained from the publicly available NCBI expressed sequence collection (dbEST), short read archive (SRA), and nucleotide database (nr), which were quality trimmed, and assembled into contigs. These sequences originated from cDNA libraries for dolphin peripheral blood leukocytes (PBL), liver, kidney, spleen, muscle and skin, and individual cloned genes from lung. The sequence set was annotated using Blast2GO [30 ] to obtain BLASTx homology (expect value cutoff E≤10^-3) gene ontology (GO) terms, and enzyme codes to determine metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Sixty-nucleotide probes designed by eArray (Agilent) were printed in a 4x44K format. Of the 24,418 sequences represented on the microarray, 7281 were fully annotated.

Custom microarrays (8 microarrays per slide) based on the published C. diphtheriae NCTC 13129 genome [39 (link)] were designed using Agilent’s eArray platform. The Agilent One-Color Microarray-Based Exon Analysis, Version 2.0 protocol was followed as per manufacturer directions. The random oligo primer mix was used for cDNA synthesis and Cyanine 3-CTP was used for subsequent cRNA synthesis. Total RNA collected from 3 independent biological replicates of each strain and condition was used for the synthesis of cRNA. Each sample was processed and run in individual wells. Arrays were washed as directed and scanned using an Agilent G2505C Microarray Scanner. For each well, the median of the gProcessedSignal for each gene probe was normalized by the mean of the entire sample set. Normalized values were log2 transformed and subjected to the J5 test using the CBER High-Performance Integrated Virtual Environment (HIVE) [40 (link)]. Genes with a log2 fold change greater than 1 and a J5 test value greater than 2 were considered significantly regulated. The microarray data and design are available at the Gene Expression Omnibus database (Accession: GSE131485).

To design the capture probe baits and prepare the SureSelect reagents, 170 kb human genomic sequence from 526 target regions were submitted to the Agilent eArray platform and manufactured by Agilent.
Peripheral blood was collected from 47 FNMTCs patients and 16 SNMTCs respectively. DNA was extracted from peripheral blood leukocytes using standard protocols. DNA of the 63 samples were extracted (QIAamp DNA blood mini kit), and the concentration of the DNA samples were measured by Qubit dsDNA assay. The gDNA quality was then assessed to make sure A260/A280 is within the range of 1.8 to 2.0. Shearing fragmentation by sonication (covaris M220) was then conducted, followed by end repair, phosphorylation and adaptor ligation. Fragments of size 200–400 bp were selected by bead (Agencourt AMPure XP Kit), followed by hybridization with the capture probes baits, hybrid selection with magnetic beads, and PCR amplification. A bioanalyzer high sensitivity DNA assay was then used to assess the quality and size range. Indexed samples were pooled to be loaded onto the flow cells for sequencing on a Miseq (Illumina, Inc., USA) with 150-cycle pair-end reads.

Probes were designed to capture open reading frame (ORF), 3′UTR, 5′UTR, and 2KB of upstream promotor of selected genes on Agilent eArray platform against hg19 human genome. Genomic DNA from patients were sonicated into fragments. A DNA library from an individual was barcoded and used for generating the library for paired-end sequencing according to manufacturers' instruction for SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). Reads were aligned in the reference genome with Burrows–Wheeler Aligner after filtering low quality reads. Single-nucleotide variants were then identified according to a previous report (15 (link)).

The capture probe baits were designed to cover 140kb human genomic loci from 186 target regions, including selected exons and introns from 18 genes (BRAF, NRAS, HRAS, KRAS, RET, NTRK1, ETV6, ALK, PPARG, TERT, EIF1AX, PTEN, AKT1, PIK3CA, TP53, CTNNB1, TSHR, GNAS) [12 (link)], so that we were able to detect all point mutation, indel, and CNV events as well as fusion events of important genes with any partner. The SureSelect reagents were prepared using the Agilent eArray platform and the probes were manufactured by Agilent.