Dataset: Peru Margin SAGs
Deployment: JRES-201

A frozen deep-sea sediment sample of the Peruvian Margin drill site 1230 (ODP 201), collected 7.3 meters below seafloor (mbsf) and stored at −80 degrees C without glycerol preservation for 8 years, was used for single cell genome analysis. Physical isolation of the single cells was performed by Fluorescent Activated Cell Sorting in two 384-well plates (630 single cells, 6 positive controls and 132 negative controls). The sample processing was performed at the Bigelow Laboratory Single Cell Genomics Center. Single cells were lysed, and the DNA was amplified by MDA. In all, 250 wells showed good amplification with a Cp value of <10 h (∼40%). DNA was screened with broad eubacterial (27F-M13: 5'-AGRGTTYGATYMTGGCTCAG-3'/907R_degen-M13: 5'-CCGTCAATTCMTTTRAGTTT-3') and archaeal (Arc_344F-M13: 5'-ACGGGGYGCAGCAGGCGCGA-3'/Arch_915R-M13R: 5'-GTGCTCCCCCGCCAATTCCT-3') 16S rRNA primers and Sanger sequenced. Analysis with the RDB (Ribosomal Database) yielded 33 hits (5.2% of all single cells sorted, 13.2% of successful MDA reactions), including three Chloroflexi single cells that showed a 16S rRNA sequence by Sanger most similar to Dehalogenimonas. The first MDA products yielded 500–900 ng of DNA after clean up with the QIAamp DNA kit (Qiagen). The first MDA products of the three single cells were re-amplified in a second MDA. To avoid additional bias, the second MDA was performed in four separate reactions that were subsequently combined at the end.

The first MDA products of the single cells were sequenced separately on an Illumina HighSeq platform (San Diego, CA, USA) using Nextera library preparation with an average yield of 15 000 Mb and 150 000 000 reads with 2 × 100 bp read length. The second MDA products were sequenced using the PacBio RSMagbead CLR sequencing technique (Menlo Park, CA, USA), resulting in a mean read length of over 2.5 Kb and ∼100 Mb raw sequence data. Sequencing was carried out according to the manufacturer's instructions and resulted in 12 Mb raw sequence data for single cell 1 and 190 Mb for single cells 2 and 3.