High sensitivity kit

Molecular analyses were performed on patients complying clinical inclusion criteria and having adequate plasma DNA available.
cfDNA was extracted from 2 to 5 mL of plasma using the AVENIO cfDNA Isolation Kit (Roche Diagnostics Spa, Monza, Italia) and eluted into 60 μL of Elution Buffer, according to the manufacturer’s instructions. cfDNA was quantified using the QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA), and cfDNA quality was assessed by Agilent Bioanalyzer using a High Sensitivity kit (Agilent Technologies, Palo Alto, CA). The extracted cfDNA was stored at −20 °C until analysis.

Total RNA quality and quantity were assessed using Agilent 2100 Bioanalyser and an RNA Nano 6000 Kit (Agilent Technologies). Sequencing libraries were prepared with 100–900 ng of total RNA with an RNA integrity number value >8 using the Illumina® TruSeq® Stranded Total RNA with Ribo-Zero Gold^TM Kit (Illumina Inc.). The steps included ribosomal RNA depletion and cleanup, RNA fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, adenylation of 3′ ends, adapter ligation and PCR amplification (12 cycles). The manufacturer’s instructions were followed except for the cleanup after the ribozero depletion step where Ampure®XP beads (Beckman Coulter) and 80% ethanol were used. Libraries were validated using the Agilent 2100 Bioanalyser and a High-Sensitivity Kit (Agilent Technologies) to ascertain the insert size, and the Qubit® (Life Technologies) was used to perform the fluorometric quantitation. Following validation, the libraries were normalised to 4 nM, pooled together and clustered on the cBot™2 following the manufacturer’s recommendations. The pool was then sequenced using a 75-base paired-end (2 × 75 bp PE) dual index read format on the Illumina® HiSeq4000 according to the manufacturer’s instructions.

Amplification of 2 ng ChIP DNA was essentially performed as described by the manufacturer (NEB, cat#E6240S), with the use of precast 2% SYBR agarose gels (Invitrogen, cat#G5218-02) and excision of band size 175–400 bp. Key modifications consisted of a 30 minute ligation step, 30 minute gel solubilization at 37°C of excised gel fragments, and a prolonged, double run-through elution step (each three minutes) with preheated (55°C) elution buffer for all column purifications (Qiagen, cats#28104,28704,28004) to ensure robust recovery. Amplification of picogram precipitated DNA was performed by adding carrier up to a total of 500 pg, 1000 pg or 2 ng as indicated using purified, chromosomal E. coli DNA, sonicated to a size distribution of 200–500 bp (Diagenode current protocols). All steps were performed in Lo-Bind tubes (Eppendorf, cat#525-0130). Libraries were generated for duplex or triplex sequencing using a NEB kit (cat#E7335S), and size distributions assessed by Bioanalyzer (Agilent, High Sensitivity kit, cat#5067-4626), (Additional file 7: Figure S5 and Additional file 10: Figure S7). Full protocol included in additional files (Additional file 8: Additional Protocols and Buffer Recipes).

Isolates were transported to the Molecular Diagnostics group at the Austrian Institute of Technology where they were checked for viability at arrival. Fifteen isolates were not viable after transportation, and sequencing failed in three isolates. After a viability check of all isolates, genomic DNA was extracted with the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed using the Ion Torrent PGM platform using 400 bp read chemistry. Sequencing was performed according to the protocol recommended by Life Technologies. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies, Carlsbad, CA, USA) and its size selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analysed using the High Sensitivity Kit (Agilent, Santa Clara, Santa Clara, CA, USA). Further sample processing was performed using the Ion OneTouch Kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). Raw reads were assembled de novo using Assembler SPAdes software [7 (link)]. The genome was annotated using the RAST (Rapid Annotations using Subsystems Technology) database [8 (link),9 (link)].

To eliminate PCR artifacts and false positives, the GeneRead DNA FFPE kit was used to extract DNA. The DNA extracted from FFPE was used for multiplex PCR of a panel covering 18–21 exons in the EGFR gene. Fragment libraries were constructed using DNA fragmentation, barcode and adaptor ligation, and library amplification, according to the manufacturer's instructions, as stipulated in the Ion Xpress Plus Fragment Library Kit (Life Technologies, Grand Island, NY, USA). Size distribution of the DNA fragments was analyzed using the Agilent Bioanalyzer with the High Sensitivity Kit (Agilent, Santa Clara, CA, USA). Template preparation, emulsion PCR, and Ion Sphere Particle (ISP) enrichment was performed using the Ion PGM Template OT2 200 Kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. The ISPs were loaded onto a 314 chip and sequenced using an Ion PGM Sequencing 200 Kit v2 (Life Technologies, Grand Island, NY, USA).