Ion 318 chip kit v2

V1–V2 regions of 16S rRNA gene were amplified using the widely used primer pair F27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and R338 (5′-TGCTGCCTCCCGTAGGAGT-3′). PCR mixture (50 uL) contained 5 µl of DNA template (~5 ng), 5 µl of 10× AccuPrime™ PCR Buffer II, 0.2 µM of each primer, and 1 U of AccuPrime™ Taq DNA Polymerase High Fidelity (Life Technologies). The PCR thermal profile consisted of an initial denaturation for 2 min at 94°C, followed by 30 cycles for 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and a final step for 7 min at 72°C. To assess possible reagent contamination, each PCR reaction included a no template control sample, which did not amplify. For each amplicon, quality and quantity were assessed using Agilent Bioanalyzer 2100 and Qubit™ fluorometer. Both primers included sequencing adaptors at the 5′ end, and forward primers were tagged with different barcodes to pool samples in the same sequencing reaction. Each pool contained 8 barcoded samples. A total of 9 pools were sequenced on an Ion Torrent Personal Genome Machine (PGM) with the Ion 318 Chip Kit v2 and the Ion PGM™ Sequencing 400 Kit (Life Technologies) under manufacturer’s conditions. The raw sequences have been deposited in NCBI under the Bioproject accession number PRJNA357691.

The Ion AmpliSeq technology (Life Technologies Ltd., Paisley, UK) was used to design a panel of 72 genes (Supplementary Table  2) associated with the following IRD forms: RP, LCA, STGD, BMD, and USH [RetNet, https://sph.uth.edu/retnet/]. The Ion AmpliSeq Designer tool (https://www.ampliseq.com/browse.action) generated an optimized primers design encompassing the coding DNA sequence of the selected genes, for a total of 1.649 amplicons divided into two pools to optimize coverage and multiplex PCR conditions. Libraries were prepared using the Ion AmpliSeq Library Kit 2.0 starting from 15 ng of gDNA/pool according to manufacturer's recommendations. Template preparation was performed using an Ion OneTouch™ 2 System following the latest version of the manufacturer's manuals. The template positive Ion Sphere Particles (ISPs+) were sequenced on an Ion Torrent Personal Genome Machine® (PGM) System (Life Technologies Ltd., Paisley, UK) using the Ion 318™ Chip kit v2 following the Ion PGM Sequencing 200 Kit v2 manual.

Total RNA samples were extracted from the cultures with the use of the RiboPure Bacteria kit (Ambion, Lithuania) as instructed by the developer’s guidelines. Afterward, the quantity and quality of the extracted RNA were verified with the use of the NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) at optical wavelengths of 260 and 280 nm. Purification of the total RNA from 16S and 23S rRNA was carried out using the MICROBExpress bacterial mRNA purification kit (Ambion, Lithuania) following the developer’s recommendations. Thereafter, the effectiveness of sample purification was determined on the Bioanalyzer 2100 (Agilent, Germany) with the RNA 6000 Nano LabChip kit (Agilent Technologies, Lithuania).
The library preparation from the extracted RNA samples included an enzymatic fragmentation step with the use of the Ion Total RNA Seq kit V2. Thereafter, the Ion Xpress RNA-Seq Barcode 01-16 kit was used for RNA fragment barcoding. cDNA sequencing was then performed using the Ion 318 Chip kit V2 on the Ion Torrent PGM sequencer (Life Technologies, USA).

The V4 region of the 16S rRNA gene was amplified with the widely used primer pair F515 (5′- GTGYCAGCMGCCGCGGTAA-3′) and R806 (5′-GGACTACNVGGGTWTCTAAT-3′). Both primers included sequencing adaptors at the 5′ end and forward primers were tagged with different barcodes to pool samples in the same sequencing reaction. The PCR mixture (25 μL) contained 2 μL of DNA template (5 ng), 5 μL of 5× Phusion^® High-Fidelity Buffer, 2.5 μL of dNTPs (2 mM), 0.2 μM of each primer and 0.5 U of Phusion^® Hot Start II Taq Polymerase (Thermo Fisher, Waltham, MA, USA).
The PCR thermal profile consisted of an initial denaturation at 98 °C for 30 s, followed by 30 cycles at 98 °C for 15 s, 55 °C for 15 s, 72 °C at 20 s and a final step at 72 °C for 7 min. To assess possible reagent contamination, each PCR reaction included a no template control (NTC) sample. For each amplicon, the quality and quantity were assessed with an Agilent 2100 Bioanalyzer and a Qubit^TM fluorometer, respectively.
Each sequencing pool included 40 barcoded samples that were sequenced on an Ion Torrent Personal Genome Machine (PGM) with the Ion 318 Chip Kit v2 and the Ion PGM™ Sequencing 400 Kit (Life Technologies, Carlsbad, CA, USA), in accordance with manufacturer’s instructions.

Samples with specific barcodes were mixed, and equal mass amounts of each 1-100 barcoded amplicons quantified by gel densitometry were pooled. Amplicons size in the library was verified using the Bioanalyzer equipment. For template preparation, emulsion PCR spheres with Ion Sphere™ Quality Control Kit (Cat# 4468656 Life Technologies^®, Thermo Fisher Scientific^®) were conditioned, and massive PCR reactions were implemented in the Thermocycler system. Likewise, the spheres’ quality was verified with the sample sequenced using Ion OneTouch™ system equipment (Cat# 4474779 Thermo Fisher Scientific^®) and Qubit 2.0 fluorometer Massive sequencing (Cat# Q33327 Thermo Fisher Scientific^®). Finally, samples’ sequencing was performed by Ion Torrent PGM™ Sequencer equipment (Cat# A25511 Thermo Fisher Scientific^®), using Ion 318 Chip Kit v2 (Life Technologies^®, Thermo Fisher Scientific^®), as previously described (6 (link)).

The WGA products from DID and TUM1 isolates were used to generate libraries and sequenced on a PGM™, Ion Torrent (Life Technologies). Briefly, fragmented DNA were ligated with sequencing barcoded adapters using Ion-Xpress barcode adapters 1–16 kit (Ion Torrent, Life Technologies). A DNA size selection was performed using E-gel size select 2% (Invitrogen, Carlsbad, USA) to retrieve fragments around 450 bp and each library was monitored using HS kit. Both libraries were equimolarly pooled then adjusted to 25 pM. Indexed libraries were clonally amplified with Ion PGM™ Template OT2 400 Kit and the Ion OneTouch™ ES Instrument (Ion Torrent, Life Technologies) according to supplier recommendations to obtain an enrichment in template-positive Ion PGM™ Template Ion Sphere™ Particles (ISP). Then, 30 µL of ISP suspension (i.e. template for DNA sequencing) were introduced in Ion 318™ Chip Kit v2 (Ion Torrent, Life Technologies) to proceed to high throughput sequencing using PGM Ion Torrent Benchtop sequencer and Ion PGM™ Sequencing 400 Kit (Ion Torrent, Life Technologies). All PGM quality-approved, trimmed, and filtered data obtained by using CLC Assembly Cell 4.1.0 were exported as SFF files.
The WGA products from CHR isolate were fragmented, ligated to Illumina adapters and the library was sequenced on a HiSeq. 2500 platform (Illumina) (2 × 150-bp).

We prepared sequence library using Ion Total RNA-Seq Kit v2 (Ion Torrent, Life Technologies, Carlsbad, CA) according to the manufacturer’s protocols except that reverse-transcribed cDNA was gel-purified to remove excess primer dimers. The region (approximately 63 nt) was excised and recovered by the same protocols as described above. Sequencing was carried out using Ion PGM Template OT2 200 Kit, Ion PGM sequencer, Ion PGM Sequencing 200 Kit v2, and Ion 318 Chip Kit v2, according to the manufacturer’s instructions (Ion Torrent). Base-calling and alignment were conducted using the built-in Ion Torrent software (V4.0.1) and the reads shorter than 24 nt were removed. Sequenced reads were mapped and aligned to coding DNA sequences extended to 25 nt upstream of translational start site of each gene, retrieved from GRCm38 using Biomart MartView (http://www.biomart.org/biomart/martview) [14 ]. As for reads with multiple hit, the best hit was aligned. All the figures and statistics below were created by the R software, if not otherwise specified.