Picogreen fluorescence assay

The University of California Microbiome Core performed nucleic acid extractions using previously published protocols (D’Amato et al. 2020 (link)). Briefly, extractions and purifications were performed using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (Thermo Fisher Scientific, USA) and automated on KingFisher Flex robots (Thermo Fisher Scientific, USA). Blank controls and mock communities (Zymo Research Corporation, USA) were carried through all downstream processing steps. Input DNA was quantified using a PicoGreen fluorescence assay (Thermo Fisher Scientific, USA) and metagenomic libraries were prepared with Illumina DNA Prep kits (Illumina Incorporated, USA) following the manufacturer’s instructions and automated on epMotion automated liquid handlers (Eppendorf, Germany). Sequencing was performed on the Illumina NovaSeq 6000 sequencing platform with paired-end 150 base pair cycles at the Institute for Genomic Medicine (IGM) at the University of California San Diego.

5 ml of whole blood was collected by ethylenediamine tetraacetic acid (EDTA) blood collection tubes then transported at ambient temperature to Nanjing Shihe Jiyin Biotech Inc. (Nanjing, China) no more than 72 h. Blood was centrifuged at 1800 × g for 10 minutes at 4°C to remove blood cells. Then, the supernatant was centrifuged at 16000 × g for 10 minutes at 4°C to remove any remaining cells. Circulating tumor DNA was extracted from 2 ml plasma, by digestion in 100 μl proteinase K buffer for 10 min at 37°C followed by purification with the NucleoSpin Plasma XS kit with modified protocols. The purified ctDNA is quantified by a Picogreen fluorescence assay using the provided lambda DNA standards (Thermo Fisher Scientific, Waltham, MA, USA).

We sequenced 300 genes that were associated with retinal disease for each patient using a capture panel that our group previously developed (Supplementary Table S1). The panel also includes the frequent pathogenic intronic variant c.2992+1655A>G that has been established to create a donor splice site within intron 26 of CEP290, causing the inclusion of a cryptic exon and a premature stop codon [17 (link)].
For capture sequencing, we generated paired-end libraries, according to the manufacturer’s protocol. We began by shearing 1 ug of genomic DNA into 300–500 base pair (bp) fragments and by adding an adenosine base to the 3′ end using the Klenow exonuclease. Y-shaped index adaptors were ligated to the fragments and 8–10 cycles of PCR amplification were performed for each sample. The DNA libraries were quantified using the PicoGreen fluorescence assay (ThermoFisher Scientific) and 50 ng of the generated libraries were used for each capture reaction. NimbleGen SeqCap EZ (Roche, Pleasanton, CA, USA) Hybridization and Wash kits were used for washing and recovering captured DNA. The captured DNA was quantified and was sent for sequencing using an Illumina HiSeq 2000 machine, following the manufacturer’s protocol. Illumina sequencing was performed at Baylor College of Medicine’s Human Genome Sequencing Center (Houston, TX, USA).