The provided definitions can be highly advantageous for professionals who are associated with or working in the field of adeno-associated virus (AAV) gene therapy research and development. AAV has become one of the most promising and widely used vectors for gene therapy due to its non-pathogenic nature, and these definitions will assist in better understanding the fundamentals of AAV gene therapy R&D.
A file format for sequencing reads aligned to a reference genome, or unaligned (uBAM), including quality metrics. Stands for “Binary Alignment/Map” format; a compressed form of the plain-text “Sequence Alignment/Map” format (SAM).
The BED (Browser Extensible Data) file format includes information about sequences that can be visualized in a genome browser; a feature called an annotation track.14 BED files are tabs-delimited and include 12 fields (columns) of data. The columns must be consistent throughout each file’s rows to be correctly read.
A file format for sequencing reads aligned to a reference genome, including quality metrics. Stands for “Compressed Reference-oriented Alignment Map” format; an even more compact form of the BAM format.
CSV (.csv file format) files stands for comma separated value and is a text file, where each line is a row and columns are delimited with a comma.18 It can store different types of sequencing data and can be opened using common spreadsheet programs like Microsoft Excel.
Demultiplexing is the process of sorting sequenced reads into separate files for each sample in a sequenced run.
File containing sequencing reads or nucleotides sequences. The FastA file format is the simplest way of representing nucleic acid of protein sequences using single-letter codes for nucleotides or amino acids.
File containing sequencing reads or nucleotides sequences and quality score for each base position. The result of base-calling and demultiplexing of raw data from a sequencing instrument.
A GFF (general feature format; file extension .gff2 or .gff3) describes the various sequence elements that make up a gene and is a standard way of annotating genomes.12 It defines the features present within a gene in the body of the GFF file, including transcripts, regulatory regions, untranslated regions, exons, introns, and coding sequences. As with the VCF, it uses a header region with a “##” string to include metadata.
The GTF (gene transfer format) file type shares the same format as GFF files, though it is used to define gene and transcript-related features exclusively.13 The attribute/group field, described above, is used in the GTF to include a gene_id or transcript_id value, a unique identifier for the genomic source or predicted transcript, respectively.
High-fidelity reads extracted from raw PacBio long-read sequencing data. HiFi reads are produced using circular consensus sequencing (CCS) mode on PacBio long-read systems. HiFi reads provide base-level resolution with 99.9% single-molecule read accuracy.
JSON (JavaScript Object Notation) is a common file format for many other industries, but is used in a growing number of bioinformatics applications.
Library preparation is the first step in sequencing. Before samples can be sequenced, nucleic acids must be isolated, fragmented, end-repaired, and covalently linked to adapters using ligation or tagmentation methods.
MAP (.map file extension) is a file format that accompanies the PED file format when using the PLINK program.17 It contains variant information.
PDB file formats contain atomic coordinates and are used for storing 3D protein structures by the Protein Data Bank. These PDB files can be opened in a program called pyMOL to visualize these protein structures. For more information on the format, see the full guide.15
PED (.ped file extension) is a file format for pedigree analysis, which creates a familial relationship between different samples16 It’s used with the PLINK command-line bioinformatics program.
The first phase of a typical workflow to analyze sequencing datasets, in which raw, multiplexed sequencing instrument data is converted into nucleotide-level base calls and quality scores and separated into distinct samples (demultiplexed).
Contiguous nucleotides sequences detected from a DNA or RNA library by a sequencing system, usually together with per-base quality scores.
A text-based file format for sequencing reads aligned to a reference genome, or sometimes unaligned, including quality metrics. Stands for “Sequence Alignment/Map” format. Compressed versions of this form include BAM and CRAM.
The second phase of a typical workflow to analyze genomic variants, in which each read set (usually from a FASTQ file or uBAM file) is filtered for quality and aligned to a reference genome.
A physical lab machine that automates the process of detecting sequences of individual nucleotide bases from a given DNA or RNA library.
In PacBio single-molecule, real-time (SMRT) sequencing technology, each polymerase read is partitioned to form one or more subreads, which contain sequence from a single pass of a polymerase on a single strand of an insert within a SMRTbell™ template and no adapter sequences. The subreads contain the full set of quality values and kinetic measurements. Subreads are useful for applications like de novo assembly, resequencing, base modification analysis, and so on.
The third phase of a typical workflow to analyze genomic variants, in which each variant call set is analyzed together with additional samples and/or auxiliary datasets such as functional and clinical annotations.
VCF (Variant Calling Format; file extension .vcf) files store gene sequence variation, such as single nucleotide polymorphisms (SNPs), and is used in genotyping projects. It contains a header with metadata preceded by a “##” string. Best practices with VCF files recommend describing INFO, FILTER, and FORMAT entries used in the body within the header.10,11
Variant Call Format, a specification for representing genomic variant calls in a text file; or a file in that format.
On the PacBio Sequencing Machine, use the on-instrument AAV mode to generate HiFi reads for proper handling of scAAV and ssAAV structures.
One ITR is formed by two palindromic arms, called B–B′ and C–C′, embedded in a larger one, A–A′. The order of these palindromic sequences defines the flip or flop orientation of the ITR.
recalladapters provides a way to change the adapter calls in a PacBio BAM file. It accepts a subreads.bam and a scraps.bam, and emits a subreads.bam and scraps.bam.
Read consists of fragments that map to one or more "genomes" (e.g. vector and host; helper and repcap)
Read originates from the host genome that is given (e.g. hg38, CHM13).
Read originates from the repcap plasmid. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization.
Read originates from the helper plasmid. In addition to Rep and Cap, AAV requires a helper plasmid containing genes from adenovirus. These genes (E4, E2a and VA) mediate AAV replication.
full
Read consists of a DNA sequence which includes the entire ITR-to-ITR target vector sequence. Read is present in both (+) and (-) polarities.
backbone
Read consists of a fragment originating solely from the plasmid backbone sequence. Read is present in both (+) and (-) polarities.
left-partial
Read consists of a fragment of the vector originating from the upstream (left ITR) of the vector while not covering the right ITR. Read is present in both (+) and (-) polarities.
partial
Read consists of a fragment of the vector originating from within the ITR sequences. Read is present in both (+) and (-) polarities.
right-partial
Read consists of a fragment of the vector originating from the downstream (right ITR) of the vector while not covering the left ITR. Read is present in both (+) and (-) polarities.
vector+backbone
Read consists of a fragment including the vector as well as plasmid backbone sequence. Read is present in both (+) and (-) polarities.
full
Read consists of a DNA sequence which includes the entire ITR-to-ITR target vector sequence. Read is present only in either (+) or (-) polarity.
backbone
Read consists of a fragment originating solely from the plasmid backbone sequence. Read is present in both (+) or (-) polarity.
left partial
Read consists of a fragment of the vector originating from the upstream (left ITR) of the vector while not covering the right ITR. Read is present only in either (+) or (-) polarity.
right-partial
Read consists of a fragment of the vector originating from the downstream (right ITR) of the vector while not covering the left ITR. Read is present only in either (+) or (-) polarity.
partial
Read consists of a fragment of the vector originating from within the ITR sequences. Read is present only in either (+) or (-) polarity.
vector+backbone
Read consists of a fragment including the vector as well as plasmid backbone sequence. Read is present only in either (+) or (-) polarity.
Read consists of a fragment mapping to the vector but with unexpected polarities (e.g. +,-,- or +,+,+) and cannot be well-defined at the moment.
