Basespace

Paired-end alignment of sequencing data against the reference genome hg19 (GRCh 37) was performed using the Whole Genome Sequencing Application v2.0, based on the Isaac Alignment Tool (Raczy et al. 2013 (link)) within BaseSpace, a cloud-based analysis tool suit (Illumina).
Somatic single-nucleotide variant (SNV) and insertion/deletion (indel) variant calling analysis was performed using the Tumour-Normal Application v1.0, based on Strelka (Saunders et al. 2012 (link)), within BaseSpace. Calls were annotated using the Variant Effect Predictor v2.8 (McLaren et al. 2016 (link)), COSMIC v77 (Forbes et al. 2015 (link)), and 1000 Genomes v3 (The 1000 Genomes Project Consortium 2015 (link)). The SIFT (Kumar et al. 2009 (link)) and PolyPhen-2 (Adzhubei et al. 2010 (link)) algorithms were used to evaluate the impact of a mutation on protein structure or function as predicted by Ensembl (v84) (McLaren et al. 2016 (link)). All variants of interest were manually inspected using Integrative Genomics Viewer (IGV) (Robinson et al. 2011 (link)).

At-home stool collection kits (DNA Genotek; OMR-200) were shipped directly to participants and then shipped back to DNA Genotek for processing. Microbial DNA was isolated from 200 μl of homogenized fecal material using the DNeasy PowerSoil Pro extraction kit (Qiagen, Germany) with bead beating in Qiagen Powerbead Pro plates (catalog no. 19311; Qiagen, Germany). Extracted DNA was quantified using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Invitrogen, USA), and all samples passed the quality threshold of 1 ng/μl (range, 8 to 101 ng/μl).
16S amplicon sequencing was performed as described previously in reference 7 (link). In brief, the 16S V3-V4 region was amplified and sequenced with 300-bp paired-end libraries on an Illumina MiSeq. Samples were demultiplexed using Illumina Basespace (San Diego, CA), yielding the FASTQ files used in this study.
Shallow shotgun sequencing was performed with the BoosterShot service (Corebiome, USA). In brief, single-stranded 100-bp libraries were prepared using an optimized proprietary protocol of the provider (Corebiome, USA) based on the Nextera library prep kit (Illumina, USA) and sequenced on a NovaSeq (Illumina, USA) to a minimum of 2.6 million (2.6M) reads per sample (mean 3.5M, ranging from 2.6M to 4M). Demultiplexing was performed on Basespace (Illumina, USA), yielding the final FASTQ files.

RNA-Seq was performed on snap-frozen tumors from traditional and alternative treatment groups. First strand cDNA was synthesized using 1 μg of total RNA, using the Illumina TruSeq mRNA library prep kit, following the manufacturer’s recommendation. Libraries were sequenced on the Illumina NextSeq 550 system (Illumina, San Diego, CA, USA). The targeted read counts were 20–35 million total reads per sample. Raw FastQ reads files were assessed and adapter trimming processed using the RNA-Seq Alignment app (Basespace, Illumina), and reads with Phred scores > 30 were retained. The resultant quality-trimmed reads were aligned to the hg38 (GrCH38.83) build of the human genome using the STAR aligner app (Basespace, Illumina). Transcript abundance quantification was performed using Cufflinks Assembly & DE analysis apps (Basespace, Illumina).

Data sequences were uploaded onto BaseSpace (BaseSpace.illumina.com">www.BaseSpace.illumina.com) (Illumina, San Diego, CA, USA) to determine sequence run quality and run completion. De-multiplexed data were downloaded locally and uploaded onto QIIME2-2018.8. The sequences were demultiplexed and the amplicon variant sequence (ASV) table was created using DADA2 [20 (link)]. The representative sequences were then classified using VSEARCH, and ASVs were defined as sequences with at least 97% similarity with Greengenes v.13.8 database [21 (link)]. Sequences that were unassigned or identified as cyanobacteria, chloroplast, or mitochondria were removed. The ASV table was then rarefied at 6400 reads for even depth of analysis. Alpha diversity metrics calculated were Shannon’s diversity index, observed OTUs (operational taxonomic units), and Chao1. Beta diversity metrics that were estimated with unweighted and weighted UniFrac distance and visualized using principle coordinate analysis (PCoA). Nonparametric test ANOSIM (analysis of similarities) was used to compare the similarity between the bacterial composition of each treatment to the baseline control (T1) using UniFrac.

The MiSeq Sequencer System Software (Illumina) was used to filter raw sequence reads and remove low quality sequences. Adapter sequences were trimmed, and sequences were further filtered to remove short sequences using the fast QC APP in BaseSpace (Illumina). Sequences that passed the filter criteria (length of 280 bp with averaged quality score of 30) were used for further analysis.
For amplicon based HTS data analysis, the 16S Metagenomics analysis pipeline in BaseSpace (Illumina) was used to make taxonomic assignments and generate data summaries of the proportions of taxa present. The sequence database for the 16S rRNA gene target was Greengenes V13_5 (DeSantis et al., 2006 (link)). For SMS data analysis, the sequences were then assembled to each of the reference genome sequences using Geneious software v10.2.6 (Biomatters Ltd) to generate contigs. Additionally, shotgun data were analyzed using APD (Advanced Probiotics species Detection; Seol et al., 2019 (link)).

Illumina BaseSpace (BaseSpace.illumina.com">https://BaseSpace.illumina.com) bioinformatics pipeline was used for sequencing QC, FASTQ Generation, genome assembly, and identification of SARS-CoV-2 variants. Briefly, the Binary Base Call (BCL) raw sequencing files generated by Illumina MiniSeq® sequencing platforms were uploaded to the Illumina BaseSpace online portal and demultiplexed to FASTQ format using the FASTQ Generation (Version: 1.0.0.) application. The raw FASTQ files were trimmed, sorted, and checked for quality (Q > 30) using the FASTQ-QC application within the BaseSpace. QC passed FASTQ files were aligned against the SARS-CoV-2 reference genome (NCBI RefSeq NC_045512.2) using Bio-IT Processor (Version: 0x04261818). Then, DRAGEN COVID Lineage (Version: 3.5.4) application in BaseSpace was used to generate a single consensus FASTA file for all the samples sequenced on a single flow cell. Finally, single consensus FASTQ was also analyzed for lineage assignment using the web version of Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) software (https://pangolin.cog-uk.io). Only the consensus variants identified by both applications were used for further analysis.