WORKFLOW
WORKFLOW
Embedded Files
Step 1: Long read Sequencing
Step 1: Long read Sequencing
Plasmidsaurus’s Premium PCR service provides full-length, amplification-free reads using Oxford Nanopore sequencing. Users receive raw .fastq files containing all variants in a mixed pool.
Step 2: Length-based Sequence Isolation
Step 2: Length-based Sequence Isolation
To ensure high-quality downstream analysis, raw reads are filtered based on length. This step removes truncated or oversized sequences, retaining only those within a specified range that correspond to full-length plasmids.
Step 3: Demultiplexing
Step 3: Demultiplexing
Filtered reads are grouped according to barcode sequences. The user supplies a barcode, and the tool matches and bins reads accordingly, separating individual variants from the pooled input.
Step 4: Export of Demultiplexed Reads
Step 4: Export of Demultiplexed Reads
After demultiplexing, grouped reads for each barcode are exported as individual .fastq files. These files can be retained for record-keeping or used in downstream custom analysis.
Step 5: De Novo Assembly (Flye)
Step 5: De Novo Assembly (Flye)
Each set of demultiplexed reads is then processed using Flye, a long-read assembler optimized for circular DNA. Flye reconstructs a full consensus plasmid sequence for each barcode group without requiring a reference sequence.
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