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Specific-Locus Amplified Fragment Sequencing (SLAF-Seq)

This method developed independently by BMKGene, can be classified inside the reduced representation genome sequencing. It 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.

It provides a fast genotyping and sets the basis for functional gene discovery or evolutionary analysis reducing the cost per sample while maintaining the 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.

Service Details

Bioinformatics

Demo Results

Featured Publications

Workflow

The service has some pre-design in silico to guarantee the optimal enzyme selection on the preparation of the library.

Technical Scheme

Service Features

● Sequencing on NovaSeq with PE150.

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

● Independent of reference genome:

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 used to choose the optimal restriction enzyme combination for the project.

Service Advantages

● High Genetic Marker Discovery: We integrate a high-throughput double barcode system allowing for the simultaneous sequencing of large populations, and locus-specific amplification enhancing 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.

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

● Extensive Expertise: We bring 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: We 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
		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

Our bioinformatical analysis comprises:

Data QC and data trimming to remove N-rich reads, adapter reads or low quality reads.

A second quality control of the clean reads to check the base distribution, sequence quality and a data assessment, but also to check the digestion efficiency and the inserts obtained.

Once the reads are checked, there are two options:

Mapping to reference genome
Without a reference genome: clustering

After it, the analysis of SLAF tags is used to do some variant calling to help with the marker discovery: SNP, InDel, SNV, CV calling and annotation

Distribution of SLAF tags on chromosomes:

Distribution of SNPs on chromosomes:

SNP annotation

Jiang S, Li S, Luo J, Wang X and Shi C (2023) QTL mapping and transcriptome analysis of sugar content during fruit ripening of Pyrus pyrifolia. Front. Plant Sci. 14:1137104. doi: 10.3389/fpls.2023.1137104

Li, J., Zhang, Y., Ma, R., Huang, W., Hou, J., Fang, C., & Sun, L. (2022). Identification of st1 reveals a selection involving hitchhiking of seed morphology and oil content during soybean domestication. Plant Biotechnology Journal, 20(6), 1110-1121. https://doi.org/10.1111/pbi.13791

Xu, P., Zhang, X., Wang, X. et al. Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nat Genet 46, 1212–1219 (2014). https://doi.org/10.1038/ng.3098

Zhuang, W., Chen, H., Yang, M. et al. The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nat Genet 51, 865–876 (2019). https://doi.org/10.1038/s41588-019-0402-2

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