Nextera dna cd indexes

We prepared DNA using the Nextera DNA Flex Library Prep Kit and Nextera DNA CD Indexes (Illumina). Next-generation sequencing and de-multiplexing were carried out by Novogene. We used CLC Genomics Workbench (QIAGEN Bioinformatics) to map reads to the Saccharomyces cerevisiae reference genome and detect SNP alleles. A table was exported from CLC that contained the location of each identified SNP in the genome, the percent of sequencing reads containing “Y” alleles at each SNP, and the number of sequencing reads crossing each SNP (i.e., the depth of coverage). Microsoft Excel was used to plot the percent of reads that have “Y” alleles at each SNP, and to plot the number of sequencing reads crossing each SNP.

Genome sequencing analysis was conducted at the Techno Suruga Co. Ltd (Shizuoka, Japan). The genomic DNA was extracted from strain FSS-1 and prepared the sequencing library (Nextera DNA Flex Library Kit, Illumina and Nextera DNA CD Indexes, Illumina) for paired-end 2×151 bp sequencing using an iSeq 100 (Illumina). Illumina reads were trimmed to remove the adapter sequences and low-quality bases, and assembled using IDBA-UD ver 1.1.2 [54 (link)]. The quality and accuracy of the acquired genomic DNA sequence were assessed using FASTX-Toolkit ver 0.0.14 [55 ]. The magnetosome genes were checked and verified manually using blastx of the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST). The mam genes, mad genes and other predicted genes of strain FSS-1 were compared with Desulfovibrio magneticus strain RS-1^T (AP010904) [56 (link), 57 (link)]. The sequence of the MGC of strain FSS-1 has been submitted to the DDBJ/EMBL/GenBank database under the following accession number: BLTE01000001. Amino acid sequences of Mam and Mad proteins identified from strain FSS-1 were used for E-value, coverage and identity analyses using the blastx of the NCBI compared with those of all magnetotactic bacteria.

We isolated DNA from each pool of animals (2 replicates × 2 treatments × 2 sexes = 8 total pools) via the Gentra Puregene Cell Kit (Qiagen, 158767) using straightforward extensions of the manufacturer’s protocol, and the resulting DNA was quantified using a fluorometer (Qubit dsDNA BR Assay Kit, ThermoFisher, Q32853). Subsequently, we used 400-ng of each DNA sample to construct indexed sequencing libraries (Illumina DNA Prep Tagmentation, 20018705; Illumina Nextera DNA CD Indexes, 20018708). Libraries were mixed by replicate into two 4-plexes, and sequenced on an Illumina NextSeq 550 instrument. We obtained 21.2–26.0 million PE150 read pairs for each replicate 1 sample, and 31.2–37.7 million PE75 for each replicate 2 sample.

Shotgun metagenomic sequencing libraries were prepared with Illumina’s Tagmentation Protocol with the Illumina DNA Prep Kit as per the manufacturer’s instructions. DNA fragments were then barcoded with Nextera™ DNA CD Indexes (Illumina). Library concentrations were determined with PicoGreen and samples were pooled for sequencing. Pooled libraries were sequenced on a NextSeq 500 sequencing platform (Illumina).

DNA was extracted from fecal samples (30-50 mg) using ZymoBIOMICS DNA Miniprep Kit [Zymo research]. Samples underwent quality control by Qubit fluorescence analysis to determine the concentration of DNA for downstream analysis (ThermoFisher, Cat. Q32850). Libraries were prepared using the Illumina Tagmentation DNA prep streamlined library preparation protocol according to the manufacturer’s instructions with a minimum of 50 ng of DNA starting mass and 8 cycles of PCR enrichment, ending with a fragment size of 550 bp. IDT for Illumina DNA/RNA UD indexes and Nextera DNA CD indexes were used (Illumina IDT, Cat. 20027213; Illumina Nextera, Cat. 20018708).
All libraries were diluted to 15 pM in 96-plex pools and validated on 100-cycle paired-ends read Miseq V2 runs (Illumina, Cat. MS-102-2002), before shipping to the US at 4 nM for sequencing on the Novaseq 6000 in S4 mode at 96-plex in a 300-cycle paired-end reads run, with an estimated read depth of 30 Gbp per sample (Illumina, Cat. 20028312). Final loading concentration of 600 pM. All sequencing runs were performed with a spike-in of 1% PhiX control library V3 (Illumina, Cat. FC-110-3001). The sequencing mean library size was 134,629,540.5 reads [range: 10,107,679 - 396,239,822].

Genomic DNA was isolated from overnight LB cultures of LT2 and MTs strains using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA), after which 150 bp paired-end libraries were prepared using the Nextera DNA Flex library prep kit (Illumina, San Diego, CA, USA) and Nextera DNA CD indexes (set of 24 indexes) (Illumina). Sequencing was carried out with an Illumina MiniSeq sequencer using a MiniSeq Mid Output Kit (300-cycles) (Illumina, San Diego, CA, USA) and analyzed with QIAGEN CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark, https://digitalinsights.qiagen.com/). The sequencing reads of our LT2 wild-type strain were trimmed and mapped to the NCBI reference genome (LT2, NCBI accession number: NC_003197.2) to generate a reference consensus sequence of our own wild-type LT2 strain. The sequencing reads of the mutant strains were then trimmed and mapped to this newly made reference LT2 genome and analyzed for single-nucleotide polymorphisms (SNPs, via basic variant detection command), indels (InDels, via InDels, and structural variants command), and structural variants (SVs, via InDels, and structural variants command). All mutations detected by WGS were verified by Sanger sequencing.

Libraries were built by using the Illumina DNA Prep protocol and Nextera DNA CD indexes (Illumina). Libraries were sequenced at the Perinatology Research Branch, using iSeq 100 reagents (Illumina) with the iSeq 100 system (Illumina) and an output of 2 × 150-bp paired-end reads.