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slag: A program for seeded local assembly of genes in complex genomes
Charles F Crane1,2, Jill A Nemacheck1,3, Subhashree Subramanyam1,3
1USDA-Agricultural Research Service, Crop Production & Pest Control Research Unit, Purdue University campus, West Lafayette, Indiana, USA.
Molecular Ecology Resources
|January 7, 2022
Summary
Seeded Local Assembly of Genes (SLAG) enables gene and chromosomal region assembly from unassembled reads. This tool is effective for low-coverage sequencing data and provides accurate contigs for primer design, offering a robust alternative for local assemblies.
Area of Science:
- Genomics
- Bioinformatics
- Computational Biology
Background:
- Finished genome assemblies are increasingly common, but challenges remain for assembling specific genes or chromosomal regions when only unassembled sequencing reads are available.
- Existing methods may require higher sequencing coverage or struggle with complex genomic structures.
Purpose of the Study:
- To introduce Seeded Local Assembly of Genes (SLAG), a novel computational tool designed for targeted gene and chromosomal region assembly from unassembled sequencing reads.
- To evaluate the performance of SLAG across various sequencing technologies and genome complexities.
Main Methods:
- SLAG employs an iterative local assembly approach, combining read retrieval using BLAST with assembly using various assemblers (CAP3, PHRAP, SPAdes, Canu, Unicycler).
- The tool supports both nucleotide and protein target sequences.
- Read fragmentation is utilized to enable assembly of long reads at lower coverages (e.g., 5×) with assemblers like PHRAP or CAP3.
Main Results:
- SLAG successfully assembled gene sequences, extending existing sequences for four wheat genes, which were validated through cloning and Sanger sequencing.
- Contig accuracy is dependent on read length and accuracy, with pyrosequencing or Illumina reads yielding contigs suitable for PCR primer design.
- Contigs from Oxford Nanopore or pre-HiFi PacBio long reads are generally suitable for designing baiting sequences for further targeted sequencing.
- SLAG demonstrates robustness, particularly for read sets with less than 20× coverage, serving as a viable alternative to tools like atram2.
Conclusions:
- SLAG provides an effective solution for local genome assembly, particularly when complete genome sequencing is not feasible or required.
- The tool's flexibility in handling different read types and coverages makes it valuable for targeted sequencing applications.
- SLAG is a robust and accessible tool for researchers needing to assemble specific genomic regions from unassembled reads.
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