Miseq dx platform

A commercially available capture-based targeted sequencing panel (solid tumors 56 gene detection kit, Burning Rock Biotech Ltd., Guangzhou, China.) was used to identify the partner gene fused to ALK (see Additional file 2: Table S2). DNA was hybridized with capture probe baits and selected with a magnetic bead. A bioanalyzer high-sensitivity DNA assay was used to assess quality and size range. The samples were then sequenced on an Illumina MiseqDx platform with paired-end reads.

For NGS assay, six to eight formaldehyde-fixed and paraffin embedded tissue (FFPE) sections of 5- to 10-µm-thin size containing more than 50% tumor cells were used, and total DNA was extract from FFPE using a TIANamp FFPE DNA Kit (TianGen, Beijing). DNA concentration was measured using a Quantus Fluorometer (Promega, Shanghai, China). Library was prepared using a RingCap amplicon library kit for the custom-designed 11-gene panel (SpaceGen, Xiamen, China). The panel targets the whole coding regions of TP53, MLH1, MSH2, MSH6, PMS2, and EPCAM and hotspot region of PIK3CA, CTNNB1, KRAS, PTEN, and POLE exonuclease domain. MSI status was determined by the NGS method including five mononucleotide repeat MSI markers (BAT25, BAT26, NR-21, NR-24, and MONO-27) within the 11-gene panel. Sequencing was performed on a Miseq Dx platform (Illumina, USA). Raw sequencing data were trimmed and aligned to human hg19 reference genome using Trimmomatic (version 0.36) and Burrow-Wheeler Aligner (BWA) (version 0.7.17). Next, aligned reads were processed to call SNVs and small Indels using Pisces (version 5.2.9), followed by variants annotation using ANNOVAR (version 20180426). MSIsensor-pro package (version 1.2.0) was used to identify MS-stable (MSS, no MSI makers), MSI-low (MSI-L, one MSI maker), and MSI-high (MSI-H, two or more MSI makers) tumors without matched normal sample (20 (link)).

A targeted DNA-based NGS panel (YuanSu™ panel, Zhiben, Shanghai, China), including a 701-gene panel that included all coding exons of 638 key cancer-related genes and selected introns of 63 commonly rearranged genes in solid tumors, was performed [10]. The total DNA of the lesion and corresponding normal tissues was isolated from 5-μm-thick slices of FFPE samples using a DNA FFPE kit (Qiagen, Valencia, CA, USA). Fragments 200 to 400 base pairs in size were selected with beads and hybridized with the capture probe baits. Hybrid selection was subsequently performed with magnetic beads, and PCR amplification was carried out. The concentration of the DNA samples was determined using the Qubit 3.0 dsDNA assay (Thermo Fisher Scientific, Waltham, MA). Paired-end 2×150 bp sequencing was performed on the MiSeq DX platform (Illumina, San Diego, CA, USA) (15 (link)). The sequenced data were analyzed by STAR Fusion software (16 (link)).

Genomic DNA (gDNA) was extracted from the cervicovaginal swabs using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions, and stored at − 80 °C until further analysis. DNA concentration and purity were analyzed using a Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), respectively.
Library preparation was performed according to the Illumina 16S Metagenomic Sequencing Library protocol. The gDNA was amplified using primers specific for the V3–V4 region of the 16S rRNA, and a subsequent limited-cycle amplification step was performed for the added multiplexing indices and Illumina sequencing adapters. The primer sequences were as follows: Forward primer, 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3ʹ; reverse primer, 5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGG.
GTATCTAATCC-3ʹ. PCR products were then normalized and pooled using the Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and library sizes were verified using the TapeStation D1000 DNA ScreenTape (Agilent, USA). The amplified genes were sequenced using the MiSeq DX platform (Illumina, San Diego, CA, USA). Paired-end sequencing was performed using MiSeq Reagent kit V3 (2 × 300 bp/600 cycle) (Illumina, San Diego, CA, USA).

Next-generation sequencing was performed for both SNP haplotyping and mutation locus analysis, employing hundreds of primer pairs. Mutation and SNP sites were submitted to Ion Ampliseq Designer (https://www.ampliseq.com/) for primer design. Overall, 138 SNPs within 1 Mb upstream and 132 SNPs within 2 Mb downstream of the mutation gene (chr16:215400–234,700 NM_000517.4 (HBA2) and NM_000558.4 (HBA1)) were selected for NGS-based α-thalassemia SNP haplotyping. Eighty-five SNP markers located either 1 Mb upstream or downstream of the mutation gene (chr11:5246696–5,248,301 NM_000518.4 (HBB)) were selected for NGS-based β-thalassemia SNP haplotyping. Only the SNPs that were heterozygous in one parent and homozygous in their parent were considered as informative SNPs. The genomic DNA of the couple and the WGA products were amplified with these primers for haplotype construction. Sequencing libraries were prepared using the sequencing library kit (NEXTflex Rapid DNA-seq Kit 96rxns, BIOO), and the libraries were sequenced on an Illumina MiseqDX platform (Illumina) using a MiSeq Dx Reagent Kit V3 (Illumina). All procedures were carried out in accordance with the manufacturer’s protocol. The sequencing data were analyzed by Peking Jabrehoo Med Tech., Ltd.

Purified WGA products and gDNA were used for SNP and CNV library preparation by an NGS library preparation kit (Yikon Genomics). All operations followed the manufacturers’ instructions. The SNP library was sequenced by the MiSeq Dx platform (Illumina), and the CNV library was sequenced by the NextSeq CN500 platform (Illumina). The raw MiSeq data were automatically filtered, and FASTQ files were generated. The raw NextSeq CN500 data were filtered, and FASTQ files were generated by ChromGo software (Yikon Genomics).

Data analysis was performed using Assign^™ for TruSight^™ HLA software (version 2.1.0.943, Illumina Inc., San Diego, CA). Sequencing data was interpreted on using the IPD-IMGT/HLA database 3.42.0 [4 (link)]. We compared the genotypes obtained with next-generation sequencing with the previous results acquired with Sanger sequencing, allowing the estimative of the NGS accuracy [35 (link)]. The Assign^™ for TruSight^™ HLA software was designed for the genotyping of HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 from the fastq sequencing reads provided by the Illumina MiSeqDx platform with the Illumina Pipeline software. The methods implemented within the Assign^™ for TruSight^™ HLA software utilizes a large number of reads per sequence.