Nextera technology

According to the manufacturer’s instructions, DNA from stool samples was manually extracted using QIAmp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). The 460-nucleotide (nt) variable region (V3-V4) from the 16S rRNA gene (Primer fw: 16S_F 5ʹ-TCG TCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGC AG)-3ʹ; primer rv: 16S_R 5ʹ (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATC C)-3ʹ was amplified by quantitative polymerase chain reaction (qPCR), for each sample, as described in the MiSeq rRNA Amplicon Sequencing protocol (Illumina, San Diego, CA).
The first PCR reaction was set up using the following conditions: one step at 95°C for 3 minutes, 32 cycles at 95°C for 30 seconds, at 55°C for 30 seconds, at 72°C for 30 seconds, and a final step at 72°C for 5 minutes. DNA amplicons were cleaned up by KAPA Pure Beads (Roche Diagnostics, Mannheim, Germany). Indexed libraries were obtained by using Nextera technology (Illumina). The final library was cleaned up using AMPure XP beads and quantified using Quant-iT™ PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA).
According to the manufacturer’s specifications, samples were pooled together before the sequencing on an Illumina MiSeqTM platform (Illumina, San Diego, CA, United States) to generate paired-end reads of 300 base-length.

The V3 to V4 region of the 16S rRNA gene was amplified using the primer set S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 [23 (link)]. Indexed libraries were prepared by limited-cycle PCR using Nextera technology (Illumina, San Diego, CA, USA) and further cleaned up with VAHTS DNA Clean Beads (Vazyme, Red Maple Hi-tech Industry Park, Nanjing, China). The libraries were pooled at equimolar concentrations (4 nM), denatured, and diluted to 5 pM before loading onto the MiSeq Reagent kit V3 (Illumina). Sequencing on the MiSeq platform was performed by using a 2 × 300 bp paired-end protocol, according to the manufacturer’s instructions (Illumina). The raw sequence files were deposited in the Sequence Read Archive (SRA) under the project number PRJNA1054360.

Internal transcribed spacer 2 (ITS2) was amplified for fungal classification using the primer set ITS3: 5’-GCATCGATGAAGAACGCAGC-3’ and ITS4: 5’-TCCTCCGCTTATTGATATGC-3’ (White et al., 1990 ). The V3–V4 region of the 16S rRNA gene was amplified for bacterial classification by using the S‐D‐Bact‐0341‐b‐S‐17/S‐D‐Bact‐0785‐a‐A‐21 primer set (Klindworth et al., 2013 (link)). Indexed libraries were prepared by limited‐cycle PCR using Nextera technology (Illumina, San Diego, CA, USA) and further cleaned using VAHTS DNA Clean Beads (Vazyme, Red Maple Hi-tech Industry Park, Nanjing, PRC). Libraries were pooled at equimolar concentrations (4 nM), denatured, and diluted to 5 pM before loading onto a MiSeq Reagent Kit V2 (Illumina, San Diego, CA, USA). Sequencing on the MiSeq platform was performed using a 2 × 250 bp paired-end protocol according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).

Library preparation was performed according to Illumina 16 S Metagenomic Sequencing Library Preparation protocol (Illumina, San Diego, CA, USA). The V3-V4 hypervariable region of the 16 S rRNA gene was amplified by PCR in 50 µL final volume, containing 25 ng of microbial DNA, 2X KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), and 200 nmol/L forward 314 and reverse 785 primers carrying Illumina overhang adapter sequences [21 (link)]. The PCR thermocycle consisted of 3 min at 95 °C, then 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a final elongation step at 72 °C for 5 min [20 , 22 (link)]. Amplified products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Indexed libraries were prepared by limited-cycle PCR using Nextera technology (Illumina) and purified again as described above. The libraries were then quantified using a Qubit 3.0 fluorimeter (Invitrogen, Waltham, MA, USA), normalized to 4 nM, and pooled. Finally, the library pool was denatured with 0.2 N NaOH and diluted to 4.5 pM with a 20% PhiX control. Sequencing was performed on an Illumina MiSeq platform using a 2 × 250-bp paired-end protocol, according to the manufacturer’s instructions (Illumina).

Genomic DNA extraction from FACS‐sorted xenografted cells was performed using DNeasy Blood & Tissue Kit (Qiagen). Targeted gene mutation screening was performed as previously described.
³⁷ (link) Briefly, oligos for patient‐specific mutations were designed using Primer3 program for performing polymerase chain reaction. All mutation‐specific oligos were designed to exclusively target human DNA, ensuring the generation of products only from human DNA. Then, amplified amplicons were normalized and mixed together based on the mutations/patients. Transposon‐based Nextera technology (Illumina) was then used to prepare illumine libraries. Amplicon libraries were sequenced on the Illumina MiSeq platform. Variant Studio (Illumina) and integrated genome viewer were used to visualize VCF and BAM data files, respectively.