title: GSM8686282: RP88L6_5_ONT_AAV_direct_assembly_and_non_assembly_reads; synthetic construct; OTHER
library_name: GSM8686282
library_construction_protocol: Plasmids were purified with Zymo MidiPrep kit. AAV samples were sequenced by Plasmidsaurus. See manuscript for details on cloning of libraries p149-p154 (dually barcoded inserts flanked by AAV2 ITRs, with different insert lengths and class [homologous/non-homologous] between barcodes).  The library for barcode dictionary generation from parental backbone p146 was generated by amplifying by PCR (primers o945+o946v2 containing P5 and P7 Illumina adapters) the GFP stuffer insert flanked by the two barcodes using 5 ng starting template (50 uL reaction, Kapa Robust, standard conditions, 10 cycles) and followed by 1x ampure beads cleanup. The resulting library was sequenced using custom primers as a fraction of a NextSeq2000 P2 100 cycles run (read1: 42 cycles with o947, index1: 20 cycles with o761 Nextera_read1 [into SapI restriction site and GFP, not used], index2: 20 cycles with o948 [into GFP, not used], read2: 16 cycles with o762 Nextera_index2). Sequencing data was demultiplexed from other samples based on the first 10 indices of read1 (GATCCGTCGA) using bcl2fastq with base mask i10y*,y*,y*,y*, yielding 195.6M reads to associate barcodes from p146. BC1 (5'/left of insert) was on cycles 1 to 16 within read 2, BC2 (3'/right of insert) was on cycles 21 to 36 within read1.  For long read sequencing, inserts from the barcoded plasmids were released by MluI+NotI digestion and size selected on agarose (Zymoclean kit). Size selected inserts were pooled and submitted to Plasmidsaurus for a custom Oxford Nanopore sequencing project.  AAV preps were submitted to Plasmidsaurus for direct sequencing (which involves an annealing step, end-repair and adapter ligation) with Oxford Nanopore.