Biorobot ez1

Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, Les Ulis, France) with the EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired end (Illumina, San Diego, CA, USA), as previously described [23 (link)]. In order to improve the genome sequence, an Oxford Nanopore approach was performed on 1D genomic DNA sequencing for the MinIon device using an SQK-LSK109 kit. The library was constructed from 1 µg genomic DNA without fragmentation and end repair. Adapters were ligated to both ends of genomic DNA. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the library was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA). A total of 1047 active pores were detected for the sequencing and the WIMP workflow was chosen for bioinformatic analysis in real time. After 1 h of run time and end life of the flowcell, 617,960 reads were generated as raw data.

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

Each patient donated a sample of blood (~4 mL) for research, collected to EDTA-Vacutainer tubes, at the same time of blood collection for routine analytic follow-up. The collected blood was centrifuged to obtain the buffy coat, which was used to isolate and purify DNA by the EZ1 DNA Blood kit in the EZ1 BioRobot (QIAgen, Hilden, Germany). The selected SNPs were genotyped using the Sequenom Mass ARRAY matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers were designed using semi-automated Assay Design 3.1 Software (Sequenom). The global genotyping success rate was 99.85% with 100% replication agreement among one third of the samples.

Overnight cultures of E. coli were grown in BHI broth and genomic DNA extraction was extracted using the Qiagen EZ1 biorobot using the EZ1 DNA tissue kit. Sequencing libraries were prepared using Illumina Nextera XT library preparation kit and were sequenced on Illumina MiSeq using the MiSeq 600 cycle reagent version 3 kit according to standard manufacturer’s protocols.

As described previously for chikungunya virus WT_IC virus was competed with NS5_Reenc_IC virus [27 (link)]: five initial TCID50 ratios (WT_IC/NS5_Reenc_IC virus: 1/99, 20/80, 50/50, 80/20, 99/1) were used to infect a 25cm² culture flask of confluent BHK21 cells at a calculated moi of 0.5. Cells were washed twice with HBSS and then incubated for 48h after addition of 7mL of medium. Recovered infectious cell supernatant was then sequentially passaged 10 times in the same manner with the clarified cell supernatant medium from the previous passage. At each passage, a calculated moi of 1 was used. Aliquots of cell supernatant from each passage were clarified by centrifugation and stored at -80°C. Viral RNA was extracted from clarified culture supernatant medium using the EZ1 Virus Mini Kit v2 on the EZ1 Biorobot (both from Qiagen). Using two specific quantitative real time RT-PCR assays targeting the re-encoded NS5 coding region (see the quantitative real time RT_PCR assays section for more details), the amount of viral RNA was assessed for each virus (WT_IC and NS5_Reenc_IC) and the ratio of the two values (WT_IC/NS5_Reenc_IC) was calculated.

The cell supernatant media were clarified and treated with viral lysis buffer AVL (Qiagen). Following RNA extraction using an EZ1 mini virus 2.0 kit and an EZ1 Biorobot (both from Qiagen), a set of specific primer pairs was used to generate overlapping amplicons covering the full-length genome (excluding the 41 nt upstream polyA tail) with the RT-PCR Taq HIFI System (Invitrogen). The amplified DNA was analyzed using an Ion PGM Sequencer^{23 (link)} (Life Technologies) to perform complete genome sequencing. The read sequences obtained were analyzed with CLC Genomics Workbench 6 software. They were trimmed, first using the quality score, then by removing the primers used during the amplification and finally at the 5′ and 3′ termini by systematically removing 6 nucleotides. Reads with a length greater than 29 nucleotides were used and mapped to the original genome sequence, which was used as a reference. To assess the intra-population genetic diversity, the mutation frequencies for each position were calculated as the number of reads with a mutation compared to the reference divided by the total number of reads at that site. Only substitutions with a frequency of at least 1% were taken into account for the analysis (Supplementary Table S1).

Colony mass was collected from plates and used for DNA extraction with the Qiagen blood and tissue kit, automated on a Qiagen EZ1 Biorobot. Extracted DNA was used for Illumina Nextera XT library preparation according to the manufacturer’s instructions and sequenced to >50x coverage paired-end 2 × 250bp on an Illumina MiSeq instrument. Sequence data were uploaded to the European Nucleotide Archive (ebi.ac.uk/ena) and are publicly available under project accession number PRJEB34530. MLST for Yersinia [19] was performed using tools at EnteroBase [20], whereas Campylobacter and E. coli MLST was performed using the Center for genomic epidemiology (CGE) MLST tool [21]. New loci and sequence types found were submitted to the databases. Reference data for C. jejuni MLST was downloaded from pubMLST including all available profiles from isolates from humans (n = 457), chickens (n = 415) and wild birds (n = 446, of which 6 from corvids). Sequence-based E. coli serotyping was performed using the CGE SerotypeFinder tool [22]. In silico PCR was used to determine the presence of Yersinia virulence genes ail, ystA, ystB, inv, yadA, virF/lcrF [23] and irp2 [24], on assemblies created using SPAdes 3.5.0 [25] run with the – careful flag. Resistance genes were detected using ARIBA and the ResFinder database (2019-03-29) [26].