title: WGA of Candidatus Puniceispirillaceae bacterium DNA microencapsulated from seawater
design: In brief: Gulf of Maine seawater was partitioned into SemiPermeable Capsules (SPCs), in order to encapsulate particles under ~70 m in diameter (many containing DNA), which were then subject to whole genome amplification and Illumina sequencing. In depth: SPCs containing cryopreserved seawater were generated using the FLUX SPC generator (CHP-SPC1, Atrandi Biosciences) and the corresponding SPC reagent kit (CKP-G34, Atrandi Biosciences). The Working Core (WCS) and Working Shell (WSS) Solutions were prepared following manufacturer instructions, except for dithiothreitol, which was excluded. Specifically, for each lane, 37.5 l of diluted seawater was combined with 50 l of Core Reagent and 12.5 l of Photoinitiator. Before encapsulation, all samples were passed through a 40-m-mesh-size cell strainer (Becton Dickinson) and diluted with sterile-filtered and UV-irradiated Sargasso Sea water to achieve 23% SPC occupancy, as determined through an iterative process by the microscopy of post-amplification SPCs (see below). This corresponds to 11.5% of the occupied SPCs harbouring >1 DNA-containing particle. Post encapsulation, the emulsion was transferred into a 2.0-ml microcentrifuge tube, the oil phase was aspirated from the bottom using a 1-ml pipette, and the SPCs were isolated by disrupting the emulsion with the Emulsion Breaker solution included in the kit. The recovered SPCs were then resuspended in molecular grade water containing 1 Wash Additive. Alkaline lysis and DNA amplification in SPCs were performed following the WGA-X protocol24 instead of using lysis and amplification reagents provided in the kit. Briefly, SPCs were treated with 0.4 M KOH, 10 mM EDTA and 100 mM dithiothreitol for a 10-min incubation at 20 C, followed by neutralization and buffer replacement using Wash Buffer (10 mM Tris pH 7.5, 0.1% Triton X-100). The WGA-X reactions contained 0.2 U l1 Equiphi29 polymerase (Thermo Fisher), 1 Equiphi29 reaction buffer (Thermo Fisher), 0.4 mM each dNTP (New England BioLabs), 10 mM dithiothreitol (Thermo Fisher) and 40 M random heptamers with two 3-terminal phosphorothioated nucleotide bonds (Integrated DNA Technologies) (all final concentrations). These reactions were performed at 45 C for 1 h and then inactivated by a 15-min incubation at 75 C. Following the WGA-X, SPCs were washed with 1 ml wash buffer three times. To confirm DNA amplification, the SPCs were stained with 5 M SYTO-9 (Thermo Fisher) in wash buffer.  Combinatorial barcoding of WGA products within SPCs was conducted using the Single-Microbe DNA Barcoding kit (CKP-BARK1, Atrandi Biosciences). Approximately 150 l of post-WGA-X SPCs, containing ~6,000 SPCs with WGA products, were passed through DNA debranching and end preparation protocols to yield A-tailed dsDNA fragments. A 4-step, ligation-based combinatorial split-and-pool barcoding process was then employed, generating a barcode diversity of 244 (331,776) unique variants. The barcoded emSAGs were released from SPCs using the Release Reagent (Atrandi Biosciences) and purified using 0.8 Ampure XP (Beckman Coulter) paramagnetic beads. Subsequently, the next-generation sequencing libraries were prepared following the protocol of the NEB Next Ultra II FS DNA Library Prep Kit for Illumina (E7805S, NEB) using a protocol-specific ligation adapter (Atrandi Biosciences) and Unique Dual Indexing (UDI) PCR primers as directed by the Single-Microbe DNA Barcoding kit. Libraries were quantified using the Agilent High Sensitivity DNA kit (5067-4626, Agilent) and the Qubit Fluorometer with a dsDNA HS Assay kit (Q32851, Thermo Fisher). The prepared SAG libraries were loaded on the Illumina NextSeq 2000 platform using a High Output P3 kit (300 cycles) with sequencing read lengths of R1 = 151 bp, R2 = 151 bp, i7 = 8 bp and i5 = 8 bp. Read demultiplexing proceeded in two stages. In the first stage, we used bcl2fastq2 v.2.2.0 software (Illumina) with the parameter barcode-mismatches=3 to convert basecall files to pairs of fastq files and separate reads on the basis of the Illumina library indexes. The second stage used the paired fastq files as input and the Pheniqs (v.2.1.0)52 software to separate reads on the basis of the combinatorial Atrandi barcodes. To ensure demultiplexing stringency, we used a frequency threshold of 4 out of 1 million reads to exclude spurious barcode combinations and ran Pheniqs using the minimal distance decoding algorithm and distance tolerance set to allow 1 mismatch in each Atrandi barcode combination. It should be noted that an earlier version of the manual mistakenly indicated compatibility with the NEB Next Multiplex Oligos for Illumina (Dual Index Primers Set 2), NEB (P/N: E7780S) indexing primer kit, which compromised barcode-bearing fragment enrichment, resulting in only ~30% of reads displaying a recognizable cell barcode. This issue has been rectified in subsequent manual editions, ensuring reference to compatible indexing primers and achieving greater than 90% readability of cell barcodes in reads. Following demultiplexing, we performed two-stage read trimming. First, Trim Galore! (v.0.6.7)53 command trim_galoreclip_R2 45 paired -o output ${input_forward_readfile} ${input_reverse_readfile} was used to remove Atrandi barcodes. Subsequently, Trimmomatic (v.0.32)54 command trimmomatic PE -phred33 ${ input_forward_readfile } ${input_reverse_readfile } LEADING:0 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:36 was used to remove Illumina adapters and perform general quality-based trimming. Only reads containing their pairs and longer than 36 bp were retained as previously benchmarked.