454 gs junior system

Sanger sequencing was applied as previously described (16 (link)). Briefly, RNA was extracted from the CML-T1 and CML-T1/IR cells with TRIzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the complementary DNA was synthesized by M-MLV reverse transcriptase (Promega, Madison, WI, USA) using random hexamer primers (Jena Bioscience GmbH, Jena, Germany). The cDNA region encoding the kinase domain of the fused BCR-ABL was amplified using nested PCR. The resulting 914-bp amplicon was sequenced from both strands. Based on the conclusive observation of mutated BCR-ABL transcripts, we explored the analysis using next-generation deep sequencing (NGS) with IRON-II BCR-ABL plates (IRON, International Robustness of Next-Generation sequencing) on a 454 GS Junior system (Roche Applied Science, Penzberg, Germany) to reveal the presence of mutations below the detection limit of Sanger sequencing. The protocol and algorithm previously established for NGS data evaluation (17 (link)) were followed.

The extraction of total RNA from CLL samples was performed with Trizol according to the manufacturer's recommendations (Invitrogen, Life Technologies, Carlsbad, CA).
In total, 1 μg of whole extracted RNA per patient was transcribed into complementary DNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Resulting cDNA underwent polymerase chain reaction amplification of the IGVH locus using modified BIOMED-2 framework region 1 consensus primers in combination with a consensus J segment primer, as previously described.¹¹ (link) The analysis of the data was performed using the Roche (Basel, Switzerland) proprietary software package for the 454 GS Junior system (Roche). Image acquisition and treatment and signal processing were performed during the run. The bioinformatical analysis was executed using the 454 GS Junior system, as previously described.¹¹ (link)

Whole unstimulated saliva was collected from nine pSS patients (age 45–79 years) consisting of eight females and one male, and from nine healthy female controls (age 39–68 years). They all had a normal salivation rate of >1.5 ml in 15 min. All patients fulfilled the revised American European Consensus Group criteria for classification of pSS (9 (link)). DNA was extracted from the samples (200 µl volume) using the MasterPure^TM DNA Purification kit (Epicentre, Illumina Company, Madison, WI) and the final DNA was dissolved in 1×TE buffer. The 16S rRNA hypervariable region V1V2 was sequenced on a 454 GS Junior system (Roche, Branford, CT) using the primers (9 (link)) listed in Table 1. Molecular identifier (MID) tags, 10-mer, were used as sample identifiers and are listed in Supplementary Table 1. Amplification reactions were performed as described by Siddiqui et al. (10 (link)), with minor modifications as follows: the cycling program was reduced to 30 cycles and triplicate PCRs were performed for each sample. All PCR products were pooled and purified using Agencourt AMPure PCR purification (Beckman Coulter, Brea, CA). DNA quality and concentration were assessed with Bioanalyzer 2100 (Agilent, Santa Clara, CA) and Nanodrop 3300 Flurospectrometer (Thermo Scientific, Wilmington, DE).

Stool samples were collected at 3 time points: at the beginning of the pretransplantation conditioning regimen, usually 6 days before transplant (T₀); 10 days following transplant (T₁), in correspondence with the period of full aplasia and the epidemiological peak of post-transplant sepsis [23 (link)]; and 30 days after transplant (T₂) (Figure 1). The fecal microbiome was analyzed using the 454 GS Junior System (Roche Applied Science, Mannheim, Germany), using polymerase chain reaction (PCR) primers targeting the V3–V5 regions of the 16S ribosomal RNA gene. Full methods and details on the bioinformatic analysis are reported in the supplementary material.