Real time analysis v1

Data collected from NGS experiments were analyzed in order to identify single nucleotide variants and small insertions/deletions. The first steps (including base calling and demultiplexing) was performed using MiSeq provided software (Real Time Analysis RTA v.1.18.54 and Casava v.1.8.2, Illumina, Inc., San Diego, CA). FastQ files for each sample, containing mate paired-end reads after demultiplexing and adapter removal, were used as input for MiSeq pipeline. Brefly, FastQ files were processed with MiSeq Reporter v2.0.26 using the Custom Amplicon workflow. This analytical method required FastQ files, a “Manifest file” containing information about the sequences of primer pairs, the expected sequence of the amplicons and the coordinates of the reference genome (Homo sapiens, hg19, build 37.2) as input. Each read pair was aligned using the MEM algorithm of the BWA software [27 (link)]. The aligned BAM file were used as input to GATK variant caller (Genome Analysis ToolKit, v1.6) [28 (link)], thus generating a VCFv4.3 file for each sample. NGS data have been uploaded and are available at the public repository for research data Harvard Dataverse https://doi.org/10.7910/DVN/DEAEVL. All other data are within the paper and its Supporting Information files.

Genomic DNA was prepared (Truseq DNA kit) and fragmented using a Covaris LE220 focused-ultrasonicator (Covaris, Inc., Woburn, MA, USA). Sequencing libraries were prepared with KAPA Hyper Prep Kit (Roche, Basel, Switzerland). Libraries were then barcoded, pooled, and sequenced on an Illumina MiSeq with a paired-end 150 bp read configuration, generating 6.2–6.7M read pairs for each sample. On-instrument secondary analysis was performed with MiSeq Reporter Software v2.5.1 (Illumina, Inc.) using base calls and quality scores generated by Real-time Analysis (RTA) v1.18.54 (Illumina, Inc.). BWA mem 0.7.12 [121 ] was used to align reads for each sample to the C. elegans reference genome WBcel235. The resulting alignments were converted to coordinate sorted BAM format with SAMtools 1.10, and evaluated for PCR duplicates using Picard 2.7.1 [http://broadinstitute.github.io/picard]. GATK 3.7 [122 (link)] was used to further refine alignments and to call variants with the HaplotypeCaller. SnpEff 4.3t [123 (link)] was used to annotate variants according to WBcel235 reference data (snpEff_v4_3_WBcel235.86). Filtering with bcftools 1.8 [http://www.htslib.org/] selected high quality calls (QUAL > 100), homozygous alternate in one of the two samples, and at least five reads supporting each variant.

DNA was isolated using a Wizard Genomic DNA Purification Kit (Promega, Madison). The concentration of genomic DNA was measured using a Nanodrop2000 UV-Vis Spectrophotometer (Thermo scientific) and Qubit DNA assay kit. DNA integrity was evaluated by agarose gel assay (1.5%, w/v).
NGS libraries were prepared using the Illumina TruSeq Nano DNA Library Preparation Kit. Completed libraries were evaluated using a combination of Qubit dsDNA HS, Caliper LabChipGX HS DNA and Kapa Illumina Library Quantification qPCR assays. Libraries were combined in a single pool for multiplex sequencing and the pool was loaded on one standard MiSeq flow cell (v2) and sequencing performed in a 2x250 bp, paired end format using a v2, 500 cycle reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.8.4. The genomes were assembled into contiguous sequences using SPAdes version 3.9 following the manual as described previously[20 (link)], then short and low-coverage contigs were filtered out.

Here, for simplicity we use ‘microbiome’ to refer to the bacterial and archaeal community members captured by amplifying and Illumina sequencing of the 16S ribosomal RNA and DNA (rRNA gene). Library preparation and sequencing was performed by the Michigan State University Genomics Core Research Facility. A single library was prepped using the method in Kozich et al. [29 (link)]. PCR products were normalized using Invitrogen SequalPrep DNA Normalization Plates. This library was loaded onto 4 separate Illumina MiSeq V2 Standard flow cells and sequenced using 250 bp paired end format with a MiSeq V2 500 cycle reagent cartridge. Base calling was performed by the Illumina Real Time Analysis (RTA) V1.18.54.
All samples were first checked for any contaminating primer sequences using cutadapt [30 (link)], before being processed together using the USEARCH pipeline [31 (link),32 (link)]. Briefly, paired end reads were merged using -fastq_mergepairs and then dereplicated using -fastx_uniques. Reads were clustered de novo at 97% identity and then the original merged reads were mapped to the representative sequences of each cluster. Each operational taxonomic unit (OTU) was classified using SINTAX [33 (link)] and with the Silva database (v. 123, [34 (link)]).

The DNA labeled with ssARP was bound to the Dynabeads MyOne Streptavidin C1 (Invitrogen), the beads were washed with the manufacturer recommended 2× DNA binding and wash buffer (B&W buffer) and were separated from the solution on a magnetic stand (DynaMag, Invitrogen). The supernatant containing the unbound DNA was removed, the beads were washed with 1× B&W buffer, and resuspended in 1× TE. The bound DNA was released from beads by incubating with 100 mM dithiothreitol for 10 min at 37°C. Beads were placed on the magnetic stand, and the supernatant which contained the eluted DNA was collected. DNA was concentrated using ethanol precipitation.
The pulled-down DNA was used for DNA library preparation using Illumina TruSeq nano kit (Illumina). All the libraries were pooled in equimolar quantities for multiplexed sequencing and sequenced on Illumina MiSeq platform (Michigan State University). The sequencing was performed in a 2× 150 bp paired-end format using a MiSeq v2 300 cycle reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format using Illumina Bcl2fastq v2.19.1. The list of fastq files and their accession numbers are provided in Supplementary Table S3.

Data collected from NGS experiments were analyzed in order to identify single nucleotide variants and small insertions/deletions.
The first steps of bioinformatic analysis (including base calling and demultiplexing) have been performed using MiSeq provided software (Real Time Analysis RTA v.1.18.54 and Casava v.1.8.2, Illumina, Inc., San Diego, CA). FastQ files provided for each sample, containing mate paired-end reads after demultiplexing and adapter removal, were used as input for two different pipelines.