Hybrid scaffolding connects sequences by optical mapping data. Note the configuration and examine the output with visualization software for best results.
Depending on the experimental design, different pipelines may be used for one- or two-enzyme hybrid scaffolding. The latest official tools from Bionano may be found here. There are also other published tools, such as the Sewing machine pipeline.
How to configure a scaffolding run?
Similar to assembly, proper argument file settings is important.To select the right template, note for the followings:
- Platform of Bionano system (Irys/Saphyr)
- Is haplotype resolution needed
While all parameters are adjustable within the XML file, many parameters are optimized by the manufacturer, such as to suit the different optics and data nature of the two platforms. Here are the most common parameters for users to adjust:
- Enzyme: Beware to feed in the right BNX file for each enzyme correspondingly
- Genome size: for estimation of coverage of molecule support needed for trimming or extension
What are the major output files?
The major output files are the FASTA sequence files and the AGP files.
There are two versions, where on of them incorporates sequence motifs of OM signals detected within sequence gaps.
AGP is a standard file format that tells the original contig information of each resulted scaffold. More specification details can be found on the NCBI webpage
What should be performed during manual examination and curation?
This depends on the genome and data properties, such as signal density distribution, original draft genome quality, any other genome information present, as well as mapping data quality, and hence should be considered in a case-by-case basis. Examples include observation of molecule support across junctions, any translocation which may indicate possible scaffold placement, and comparison with other data, such as synteny information.
Why are there gaps of negative/ very small sizes (e.g. <500 bp) in resultant AGP files?
This may be due to overlapping ends of sequence scaffolds, where the gaps may be closed. Look out for repeated mapping patterns in sequence in silico maps, and compare with optical molecules using visualization software such as OMView, check at the sequence levels, and perform gap closing on sequence scaffolds with adjusted parameters where necessary.