Miseq system

Gene mutation analysis was performed by NGS of the DNA samples extracted from the bone marrow monocytes in patients. The NGS libraries were paired-end sequenced (2 × 150 bp) on an Illumina MiSeq System (Illumina, San Diego, CA). The mean depth of each sample was 2500 × , with an average of 98% of the target sequence covered sufficiently deep for variant calling. Detection sensitivity was ~ 5% (a mutation with 5% or more variability can be reported).
The Illumina MiSeq System (Illumina, San Diego, CA) is a high-throughput sequencing platform based on Sequencing by Synthesis (SBS) technology and sequence libraries to produce large amounts of high-quality data. Analyses were conducted of the relevant mutations of 22 genes, ASXL1, NPM1, KIT, FLT3, CEBPA, DNMT3A, IDH1/2, TET2, EZH2, RUNX1, PHF6, TP53, SF3B1, SRSF2, U2AF1, ZRSR2, NRAS, CBL, SETBP1, ETV6, and JAK2. Each mutation was analyzed in the Catalog of Somatic Mutations in Cancer databases (COMSIC; https://cancer.sanger.ac.uk/cosmic/). All bone marrow samples were collected with informed consent, and the study was reviewed and approved by the First Affiliated Hospital of Zhengzhou University College of Medicine.

Whole genome sequencing was conducted for all Xtg strains listed in Table 1. In view of the sequencing costs, homopolymer resolution, and observed GC bias in library preparations [22 (link), 23 (link)], we decided to apply the Illumina MiSeq System by sequencing (2 × 250 bp) paired-end libraries constructed with the TrueSeq DNA LT Sample Prep Kit (Illumina Inc., San Diego, United States). The Swiss strain Xtg29, which has previously been sequenced by applying the Roche 454 Genome Sequencer FLX System [12 (link)], was re-sequenced in order to prevent bias in comparative genome analysis due to deviating sequencing strategies. Genome assembly of Xtg29 was moreover facilitated by (2 × 250 bp) sequencing of an additional 8 kb mate-pair library on an Illumina MiSeq System (Illumina Inc., San Diego, United States). For the following comparative genome analysis, we used Xtg29 as our reference strain for Xtg.

For metagenomic DNA analysis, loog-pang and koji starters were extracted and purified following the instructions of PureLink^TM microbiome DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) and the samples were stored at -20 °C until use. The purified DNA concentration was determined using a Qubit fluorometer (Thermo Fisher Scientific) and gel electrophoresis was used to determine the presence of genomic DNA. The metagenomic sequencing of 16S or internal transcribed spacer (ITS) preparation followed similar standard guidelines of the 16S metagenomic sequencing library preparation protocol of the Illumina MiSeq System (Illumina, San Diego, CA, USA), but with different primers. The V3-V4 segment of bacterial 16S rRNA was amplified with forward (5’-CCTACGGGNGGCWGCAG-3’) and reverse (5’GACTACHVGGGTATCTAATCC-3’) primers. For fungi, the ITS segment was amplified with forward (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and reverse (5′-GCTGCGTTCTTCATCGATGC-3′) primers. Sequencing was performed on a MiSeq system (Illumina). For bacterial and fungal data preparation, raw sequence reads of approx. 300 bp were analysed with MiSeq reporter and the Greengenes database (20 (link)) was used to classify the genus and species of the microbiome diversity of the sample.