Read1 sequencing primer

The Illumina Read1 sequencing primer, 16 bp barcode, and 6 bp random sequence primer were connected to the GelBeads sequence. The GelBeads passes through two injection ports. The input DNA is mixed with enzymes and dNTP and other reactants after entering the first sample inlet, and the mixture is wrapped by oil drops when passing through the second sample inlet, then it is collected and placed on a special 96-well plate for the barcode library preparation. The random primers were combined with the random position of genomic DNA fragments in the oil drop reaction system for constant temperature PCR amplification. After the library preparation by adding the Illumina P5 and P7 adaptors into the amplification products, PE150 was sequenced on the Illumina platform at Beijing Novogene Co. Ltd (Beijing, China). An approximate 119.10-fold genome coverage (194.14 Gb) 10X Genomics Linked-Reads were generated for the assembly.

Genomic DNA (gDNA) with an adjusted concentration between 1.0 and 1.25 ng/µL was used to prepare the whole-genome sequencing libraries using the Chromium Genome Library and Gel Bead Kit v.2, Chromium Genome Chip Kit v.2, Chromium i7 Multiplex Kit, and Chromium controller according to the manufacturer's instructions (10X Genomics). Genomic DNA was combined with Master Mix, a library of Genome Gel Beads, and partitioning oil to create Gel Bead-in-Emulsions (GEMs) on a Chromium Genome Chip. The GEMs were isothermally amplified with primers containing an Illumina Read 1 sequencing primer, a unique 16-bp 10X barcode, and a 6-bp random primer sequence. Barcoded DNA fragments were recovered for Illumina library construction. The amount and fragment size of post-GEM DNA were quantified prior using a Bioanalyzer 2100 with an Agilent high-sensitivity DNA kit. Prior to Illumina library construction, the GEM amplification product was sheared on an E220 Focused Ultrasonicator (Covaris) to approximately 350 bp. Then, the sheared GEMs were converted to a sequencing library following the 10X standard operating procedure. The library was quantified by quantitative polymerase chain reaction (qPCR) with a Kapa Library Quant kit (Kapa Biosystems–Roche) and sequenced on a NovaSeq6000 sequencer (RRID:SCR_020150) (Illumina) with paired-end 150-bp reads.

Following a successful deep-screening display experiment, we set-up a second sequencing experiment on a fresh flow cell using the same library for resolving the CDR sequences with internal sequencing primers. CDR sequencing experiments were performed in HCS with a custom recipe that initially sequenced the N₂₈ UMI with Illumina’s Read 1 Sequencing Primer for 28 cycles, followed by denaturation of the sequencing product with FDR at 65 °C, annealing of an appropriate internal sequencing primer and sequencing enough cycles to cover the region of variability. All internal sequencing primers used in this work were ordered from IDT, HPLC purified and resuspended in IDTE at 100 µM.