Sequencing analysis v5

All collected blood samples were anticoagulants with EDTA. Genomic DNA from each enrolled patient was extracted using the DNA Isolation Kit for Blood/Bone Marrow/Tissue (Tiangen Biotech (Beijing) Co., Ltd.). DNA concentration and purity were evaluated using a NanoDrop2000 (Thermo Scientific). The primer sequences and the restriction enzymes used were provided by SINOMD Gene (Beijing) Co., Ltd. Genomic DNA was used for the cyclic condition consisted of one predenaturation step at 95 °C for 3 min initial cycle, then 45 cycles of 94 °C for 15 s, 63 °C for 1 min and extension at 72 °C for 1 min, and a final elongation step of 10 min at 72 °C. PCR products were purified with ExoSap-It (ABI PCR Product Cleanup Reagent). Single-nucleotide polymorphisms were analyzed using ABI 3500 Dx Genetic Analyzer (ABI Terminator v3.1 Cycle Sequencing kit) and Sequencing Analysis v5.4 (Life Technologies, CA, USA). We looked for the following SNPs: DPYD*2A, DPYD*5A, DPYD*9A, and GSTP1.

PCR products, obtained by RT-PCR, were purified using the QIAquick PCR Purification Kit (Qiagen NW, DE) and were subjected to bidirectional sequencing using the Big Dye-Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA), according to the manufacturer’s recommendations. Dye terminators from sequences were removed using a DyeEx-2.0 Spin Kit (Qiagen), and sequences were run on a SeqStudio Genetic Analyzer (Thermo Fischer Scientific, CA, USA). Electropherograms were analyzed using Sequencing Analysis v5.2 and Sequence Scanner v1.0 software (Thermo Fischer Scientific, CA, USA). The sequences were analyzed using the BLAST program.

PCR products, obtained by RT-PCR, were purified using the Qiaquick PCR purification Kit (Qiagen NW, DE) and bidirectionally sequenced using the Big Dye-Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA), as per the manufacturer’s recommendations. Sequences were dye terminator removed by DyeEx-2.0 spin kit (Qiagen) and run on a SeqStudio Genetic Analyzer (Thermo Fischer Scientific, Waltham, MA, USA). Electropherograms were analysed using Sequencing Analysis v5.2 and Sequence Scanner v1.0 software (Thermo Fischer Scientific, Waltham, MA, USA). The sequences were analysed using BLAST.

PCR products, obtained by RT-PCR, were purified using the QIAquick PCR Purification Kit 131 (Qiagen NW, DE) and were subjected to bidirectional sequencing using the Big Dye-Terminator v1.1 Cycle 132 Sequencing Kit (Applied Biosystems, CA, USA), according to the manufacturer’s recommendations. Dye terminators from 133 sequences were removed using a DyeEx-2.0 Spin Kit (Qiagen), and sequences were run on a SeqStudio 134 Genetic Analyzer (Thermo Fischer Scientific, CA, USA). Electropherograms were analysed using Sequencing Analysis v5.2 and Sequence Scanner v1.0 softwares (Thermo Fischer Scientific, CA, USA). The sequences were analysed using the BLAST program.

The total bacterial cell count in the samples were confirmed by the epifluorescence staining method described previously [23 (link), 24 (link)]. Bacterial DNA were extracted with sonication method and 16S rRNA genes were amplified with a universal primer set [25 ] described previously [23 (link), 24 (link)]. The PCR products were cloned into Escherichia coli using a TOPO TA cloning kit and a total of 96 colonies were randomly selected for sequencing analysis. Data analysis was performed using ‘Sequencing Analysis V.5.2’ (Applied Biosystems). Additional details of the method are described in Additional file 1: Appendix S1. The highly accurate sequences were classified into the level of genus with RDP (ribosomal database project) classifier (confidence threshold 90%) [26 (link)]. The sequences of less than 90% confidence threshold value were deemed unclassified. Genera with proportions less than 0.1% of the total number of clones were titled ‘others’. Moreover, the in-house database (Additional file 2: Appendix S2) containing 5878 16S rRNA gene sequences of type strains using the BLAST (basic local alignment search tool) algorithm was used for more detailed analysis at the level of species. All sequences were submitted to public database (DNA data bank of Japan, Accession number: LC350341-LC360093).

A panel of 32 EC tumors was selected for mutation sequencing of Hic1 and Myo1c (Additional file 1: Table S1). The Inpp5k gene was sequenced in 18 tumors. Primer pairs were designed using the Primer3 program and synthesized by a commercial supplier (SIGMA-Genosystem, Cambridge, UK). PCR primers set corresponding to the coding sequences of Hic1, Inpp5k and Myo1c genes were amplified and screened for mutations (Additional file 3: Table S2). For the Myo1c gene the promoter region was also sequenced.
PCR amplification products were purified using GFX™ PCR DNA and gel Band Purification Kit (Amersham Pharmcia Biotech, Piscataway, NJ). Using ABI PRISM® BigDye® Terminator v1.1 or 3.1 Cycle Sequencing Kit (Applied Biosystems), the purified DNA fragment were subjected to sequencing according to the protocol provided by the manufacturer. Sequencing products were separated on a denaturing polyacrylamide gel on a 3130xl Genetic Analyzer (Applied Biosystems) and analyzed using the software’s Sequencing Analysis v5.2 and SeqScape v2.5 (Applied Biosystems).

Sequences were edited using Sequencing Analysis v.5.2 (Applied Biosystems). Sequences were aligned with MUSCLE using SeaView v.4.7 [41 (link)] and Mega X [42 (link)]. Phylogenetic trees were constructed with the maximum likelihood (ML) method and the Kimura-2 (K80) evolutionary model in Mega X. To assess the ML tree reliability, bootstraps were tested with 1000 replicates. To estimate the evolutionary divergence between sequences, genetic distances were analyzed by pairwise distance (p-distance) methods [43 (link)] in Mega X. Divergence time was calculated based on previously reported estimates giving 1 million years (Myr) for 2.3% difference [44 (link), 45 (link)]. Sequences are deposited in GenBank under the following accession numbers: ITS2: N2 (MK659798), N44 (MK659797), S9 (MK659795), S33 (MK659793), NT64 (MK659796), NT101 (MK659794), KP10 (MK659792), KP72 (MK659780), 1x (MK659773), 2M (MK675654), 4M (MK675653), P1 (MK656100), C1 (MK656095) and C2 (MK656096); cox1: N2 (MK683469), N44 (MK683470), S9 (MK683476), S33 (MK683477), NT64 (MK683473), NT101 (MK683474), KP10 (MK683471), KP72 (MK683472), 1x (MK683464), 2M (MK683465), 4M (MK683466), P1 (MK683475), C1 (MK683467) and C2 (MK683468).