Reagent kit v2

The bacterial diversity in the hindgut compartments from the two millipede species was analyzed by paired-end sequencing of the V4 region of the 16S rRNA genes on an Illumina MiniSeq platform (2 × 250 cycle configuration; V2 reagent kit; Illumina) at the DNA Services Facility at the University of Illinois, Chicago, USA (Table S1), following Naqib et al. [40 (link)]. After quantifying the DNA with PicoGreen, the samples were diluted to a final concentration of 10 ng µl⁻¹. For PCR and library preparation, the primers 515F_mod and 806R_mod [41 (link)] were used to amplify the V4 region of the 16S rRNA gene. For gene quantification, the template DNA was diluted to 0.01 ng µl⁻¹, and 2 µl was used per reaction with primers 338F–805R (0.5 µM), the 516P FAM/BHQ1 probe (0.2 µM) together with the digital droplet PCR Supermix for probes (Bio-Rad), and quantified on a QX200 AutoDG Droplet Digital PCR System (ddPCR; Bio-Rad). The full protocol can be found online [42 ]. The copy numbers of 16S rRNA were normalized for 1 ng of total DNA.

Amplicon‐based microbiome analysis was done as previously described (Ring et al., 2017 (link)), and as illustrated in Figure S1. DNA was amplified using a two‐step PCR using custom 341F/806R primers targeting the V3–V4 16S regions, and three primer sets targeting the hyper‐variable regions V3–V4 of the 18S rDNA gene. Library preparation was performed by Nextera XT DNA Library Preparation (Illumina Inc., San Diego, CA, USA), and Illumina sequencing was performed on the MiSeq system (Illumina) according to the manufacturer's instructions using the V2 Reagent Kit.

RNA was extracted from each cell line using a QIAamp DNA Mini Kit (Qiagen) or an RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA (1,500 pg per cell line) was used for targeted RNA sequencing using a SureSelect RNA Human Kinome Kit (Agilent Technologies, Santa Clara, CA, USA) targeting 612 genes, including 517 protein kinases. Sequencing was performed on a MiSeq Sequencing System via a V2 Reagent Kit (Illumina, San Diego, CA, USA). Sequencing data were analyzed using the CLC Genomics Workbench (CLC bio, Aarhus, Denmark). RNA samples from each cell line were analyzed twice and averaged.

Genomic DNA of fecal and effluent samples was extracted using the FastDNA Spin kit for soil (MP Biomedicals, Illkirch, France) according to the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified with the primers 806R (5′-GGACTACHVGGGTWTCTAAT-3′) and 515F (5′-GTGCCAGCMGCCGCGGTAA-3′). Amplicons were barcoded PCR based. Library preparation and sequencing (Illumina, CA, USA) using an Illumina MiSeq flow cell with a V2 reagent kit for 2 × 250-bp paired-end Nextera chemistry supplemented with 10% PhiX were performed in collaboration with the Genetic Diversity Center (GDC; ETH Zürich, Switzerland).
Raw data obtained from 16S rRNA sequencing were processed using Cutadapt (57 (link)) and DADA2 pipeline (58 (link)) to obtain amplicon sequence variants. Taxonomy was assigned using the SILVA database (v.132) (59 (link)) (full method described in Text S1).

Nucleic acid extraction and sequencing was carried out using a modified version of a tiling amplicon-based method to enrich the culture supernatant for viral RNA, which we previously used to sequence mumps virus from a 2010 Ontario outbreak^{13 (link)}. We extracted RNA using either the QIAamp Viral RNA Mini Kit (Qiagen, Mississauga, ON) or the NucliSENS easyMAG instrument. For the initial eight samples we performed amplification of 18 overlapping amplicons, of mean length 977 bp. We optimised the protocol to reduce the number of amplicons, so for the last 19 samples we sequenced 9 amplicons of mean length 1958 bp (Supplementary Dataset 2 for primers). Amplification of the fragments in 96 well plates was performed on a SimpliAmp thermal cycler using the superscript III One Step RT-PCR system (Invitrogen,Thermo Fisher Scientific).
Amplicon fragments from individual samples were pooled together in equal amounts and cDNA concentration checked using a Qubit fluorometer. Mumps cDNA libraries were prepared with the Nextera XT kit. We checked the quality of the indexed libraries by Bioanalyzer. Sequencing on the Illumina MiSeq instrument was performed with V2 reagent kit (2 × 150 bp, Illumina Inc. San Diego, California, USA), according to the manufacturer’s instructions.

Amplicon-based microbiome analysis was done as previous described (Ring et al., 2017 (link)). Library preparation was performed by Nextera XT DNA Library Preparation (Illumina inc., San Diego, California, United States), and Illumina sequencing was performed on the Hiseq system (Illumina) according to the manufacturer’s instructions. DNA was amplified using a two-step PCR using custom 341F/806R primers targeting the V3-V4 16S regions, and three primer sets targeting the hyper-variable regions V3-V4 of the 18S rDNA gene, and amplicons were sequenced on the Illumina MiSeq (Illumina) using the V2 Reagent Kit.

DNA of a thrombosis patient was isolated from EDTA-blood using EZ1 BioRobot (Qiagen). Targeted NGS using HaloPlex Target Enrichment reagents for library preparation (Agilent Technologies, Santa Clara, CA, USA) was applied to sequence coding sequences of 15 loci involved in haemostasis (Table 1). DNA library was sequenced on a miSeq instrument with v2 reagent kit (Illumina Inc., San Diego, CA, USA), following the suppliers’ recommendations. Sequence reads were mapped to the human reference genome (GRCh37/hg 19).
Table 1

A panel of 15 loci involved in haemostasis that were sequenced by next generation sequencing in DNA from a patient with deep venous thrombosis

Gene name	Approved symbol	Chromosome location
Protein C, inactivator of coagulation factors Va and VIIIa	PROC	2q14.3
Protein S (alpha)	PROS1	3q11.1
Serpin family C member 1 (antithrombin-III)	SERPINC1	1q25.1
Von Willebrand factor	VWF	12p13.31
Coagulation factor II, thrombin	F2	11p11.2
Coagulation factor V	F5	1q24.2
Coagulation factor VII	F7	13q34
Coagulation factor VIII	F8	Xq28
Coagulation factor IX	F9	Xq27.1
Coagulation factor XI	F11	4q35.2
Fibrinogen alpha chain	FGA	4q31.3
Fibrinogen beta chain	FGB	4q31.3
Fibrinogen gamma chain	FGG	4q32.1
Vitamin K epoxide reductase complex subunit 1	VKORC1	16p11.2
Gamma-glutamyl carboxylase	GGCX	2p11.2