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Specific-Locus Amplified Fragment Sequencing (SLAF-Seq)

High-throughput genotyping, particularly on large-scale populations, is fundamental step in genetic association studies and provides a genetic basis for functional gene discovery, evolutionary analysis, etc. Instead of deep whole genome re-sequencing, Reduced Representation Genome Sequencing (RRGS) is often employed in these studies to minimize sequencing cost per sample while maintaining reasonable efficiency in genetic marker discovery. RRGS achieves this by digesting DNA with restriction enzymes and focusing on a specific fragment size range, thereby sequencing only a fraction of the genome. Among the various RRGS methodologies, Specific-Locus Amplified Fragment Sequencing (SLAF) is a customizable and high-quality approach. This method, developed independently by BMKGene, optimizes the restriction enzyme set for every project. This ensures the generation of a substantial number of SLAF tags (400-500 bps regions of the genome being sequenced) that are uniformly distributed across the genome while effectively avoiding repetitive regions, thus assuring the best genetic marker discovery.

Service Details

Bioinformatics

Demo Results

Featured Publications

Workflow

Technical Scheme

Service Features

● Sequencing on NovaSeq with PE150.

● Library preparation with double barcoding, enabling pooling of over 1000 samples.

● This technique can be used with or without a reference genome, with different bioinformatic pipelines for each case:

With reference genome: SNP and InDel discovery

Without reference genome: sample clustering and SNP discovery

● In the in silico pre-design stage multiple restriction enzyme combinations are screened to find the ones that generate a uniform distribution of SLAF tags along the genome.

● During the pre-experiment, three enzyme combinations are tested in 3 samples to generate 9 SLAF libraries, and this information is use to choose the optimal restriction enzyme combination for the project.

Service Advantages

● High Genetic Marker Discovery: Integrating a high-throughput double barcode system allows for the simultaneous sequencing of large populations, and locus-specific amplification enhances efficiency, ensuring that tag numbers meet the diverse requirements of various research questions.

● Low Dependence on the Genome: It can be applied to species with or without a reference genome.

● Flexible Scheme Design: Single-enzyme, dual-enzyme, multi-enzyme digestion, and various types of enzymes can all be selected to cater to different research goals or species. The in silico pre-design is carried out to ensure an optimal enzyme design.

● High Efficiency in Enzymatic Digestion: The conduction of an in silico pre-design and a pre-experiment assured optimal design with even distribution of SLAF tags on the chromosome (1 SLAF tag/4Kb) and reduced repetitive sequence (<5%).

● Extensive Expertise: Our team brings a wealth of experience to every project, with a track record of closing over 5000 SLAF-Seq projects on hundreds of species, including plants, mammals, birds, insects, and aquatic organisms.

● Self-developed Bioinformatic Workflow: BMKGENE developed an integrated bioinformatic workflow for SLAF-Seq to ensure the reliability and accuracy of the final output.

Service Specifications

Type of analysis	Recommended population scale	Sequencing strategy
Type of analysis	Recommended population scale	Depth of tag sequencing	Tag number
Genetic Maps	2 parents and >150 offspring	Parents: 20x WGS Offsping: 10x	Genome size: <400 Mb: WGS is recommended <1Gb: 100K tags 1-2Gb:: 200K tags >2Gb: 300K tags Max 500k tags
Genome-Wide Association Studies (GWAS)	≥200 samples	10x
Genetic Evolution	≥30 samples, with >10 samples from each subgroup	10x

Service Requirements

Concentration ≥ 5 ng/µL

Total amount ≥ 80 ng

Nanodrop OD260/280=1.6-2.5

Agarose gel: no or limited degradation or contamination

Recommended Sample Delivery

Container: 2 ml centrifuge tube

(For most of the samples, we recommend not to preserve in ethanol)

Sample labeling: Samples need to be clearly labeled and identical to submitted sample information form.

Shipment: Dry-ice: Samples need to be packed in bags first and buried in dry-ice.

Service Workflow

Sample QC

Pilot experiment

SLAF-experiment

Library Preparation

Sequencing

Data Analysis

After-sale Services

Previous: DNA/RNA Sequencing – Nanopore Sequencer

Next: Human Whole Exome Sequencing

Includes the following analysis:

Sequencing data QC
SLAF tag development

Mapping to reference genome

Without a reference genome: clustering

Analysis of SLAF tags.: statistics, distribution across the genome
Marker discovery: SNP, InDel, SNV, CV calling and annotation

Distribution of SLAF tags on chromosomes:

Distribution of SNPs on chromosomes:

SNP annotation

Year	Journal	IF	Title	Applications
2022	Nature communications	17.694	Genomic basis of the giga-chromosomes and giga-genome of tree peony Paeonia ostii	SLAF-GWAS
2015	New Phytologist	7.433	Domestication footprints anchor genomic regions of agronomic importance in soybeans	SLAF-GWAS
2022	Journal of Advanced Research	12.822	Genome-wide artificial introgressions of Gossypium barbadense into G. hirsutum reveal superior loci for simultaneous improvement of cotton fiber quality and yield traits	SLAF-Evolutionary genetics
2019	Molecular Plant	10.81	Population Genomic Analysis and De Novo Assembly Reveal the Origin of Weedy Rice as an Evolutionary Game	SLAF-Evolutionary genetics
2019	Nature Genetics	31.616	Genome sequence and genetic diversity of the common carp, Cyprinus carpio	SLAF-Linkage map
2014	Nature Genetics	25.455	The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.	SLAF-Linkage map
2022	Plant Biotechnology Journal	9.803	Identification of ST1 reveals a selection involving hitchhiking of seed morphology and oil content during soybean domestication	SLAF-Marker development
2022	International Journal of Molecular Sciences	6.208	Identification and DNA Marker Development for a Wheat-Leymus mollis 2Ns (2D) Disomic Chromosome Substitution	SLAF-Marker development

Year	Journal	IF	Title	Applications
2023	Frontiers in plant science	6.735	QTL mapping and transcriptome analysis of sugar content during fruit ripening of Pyrus pyrifolia	Genetic Map
2022	Plant Biotechnology Journal	8.154	Identification of ST1 reveals a selection involving hitchhiking of seed morphology and oil content during soybean domestication	SNP calling
2022	Frontiers in plant science	6.623	Genome-Wide Association Mapping of Hulless Barely Phenotypes in Drought Environment.	GWAS