SNP genotyping of the association panel was performed using a SLAF-seq approach17 (link). Construction of the peanut DNA libraries and Illumina sequencing of the plants were performed at Biomarker Technologies Corporation in Beijing, China. Through restriction enzymes HaeIII and Hpy166II (New England Biolabs, NEB, USA) that digest peanut genomic DNA into DNA fragments of 364–464 bp23 (link), the sequencing libraries of 202 peanut accession were constructed. The physical position of the markers were identified by aligning the sequence of a 125 bp paired-end reads attached to each marker with the ‘pseudomolecules’ genome sequences of diploid peanut (Arachis duranensis-AA and Arachis ipaensis-BB, https://www.peanutbase.org ) using local BLASTn (BLAST: Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi ). If the reads matched two or more locations in the reference genome of peanut, the markers were regarded as non-specific markers and discarded. Accurate markers were selected throught three steps. First, all candidate markers must be called in the mixed reads from parents and all the F6 samples using GATK (https://software.broadinstitute.org/gatk/ ). Second, all candidate markers must be called less than 20%. Third, some hemi-SNPs which showed polymorphism in sub-genomes were used for polymorphism markers in genetic linkage map in RIL population.
Hpy166ii
Manufactured by New England Biolabs
Sourced in United States
Hpy166II is a Type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-GTNNAC-3'. It is commonly used in molecular biology applications such as DNA cloning, restriction mapping, and DNA fingerprinting.
Automatically generated - may contain errors
Lab products found in correlation
Haeiii, by New England Biolabs (5 mentions)
Hiseq 2500 system, by Illumina (4 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Mytaq polymerase, by Meridian Bioscience (1 mentions)
Page purified, by Thermo Fisher Scientific (1 mentions)
Klenow fragment 3 5 exo, by New England Biolabs (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Hpych4iii, by New England Biolabs (1 mentions)
Cfx96 real time thermocycler, by Bio-Rad (1 mentions)
D100 screen tape, by Agilent Technologies (1 mentions)
Cac8i, by New England Biolabs (1 mentions)
9 protocols using hpy166ii
1
Genotyping Association Panel using SLAF-seq
2
Genotyping Mouse Leptin Receptor Gene
3
Optimized SLAF-seq Sequencing Protocol
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An optimized SLAF-seq strategy was utilized in this study. Two enzymes Hpy166II + Hae III (New England Biolabs, NEB, United States) were used to digest the genomic DNA from the parent and population individuals. Then, by Klenow Fragment (3′ → 5′ exo-) (NEB) and dATP at 37°C, a single nucleotide (A) overhang was ligated with the digested fragments. Subsequently, the A-tailed fragments were connected to duplex tag-labeled sequencing adapters (PAGE-purified, Life Technologies, United States) by T4 DNA ligase. Polymerase chain reaction (PCR) was carried out using dNTP, diluted digestion-ligation DNA samples, Q5® High-Fidelity DNA Polymerase and PCR primers (Forward primer: 5′- AATGATACGGCGACCACCGA-3′, reverse primer: 5′-CAAGCAGAAGACGGCATACG-3′). Next, PCR products were depurated using Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, United Kingdom) and pooled. Pooled samples were separated by 2% agarose gel electrophoresis. Fragments ranging from 364 to 464 base pairs (with indexes and adaptors) in size were excised and purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). Gel-purified products were then diluted. And pair-end sequencing (Each end 125 bp) was performed on an Illumina HiSeq 2500 system (Illumina, Inc., San Diego, CA, United States) at Beijing Biomarker Technologies Corporation.
4
SLAF-seq for Genotyping RILs
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The SLAF-seq strategy was used in this study (Biomarker Technologies, Beijing, China) (Sun et al., 2013 (link)). Genomic DNA from 164 RILs was digested with HaeIII and Hpy166II [New England Biolabs (NEB), Ipswich, MA, USA]. Subsequently, a single nucleotide (A) was added to the 3′ end of the digested fragments using Klenow Fragment (NEB) and dATP. T4 DNA ligase was used to ligate the Duplex tag-labeled sequencing adapters to distinguish them from raw sequencing data. PCR was performed using forward (5′-AATGATACGGCGACCACCGA-3′) and reverse (5′-CAAGCAGAAGACGGCATACG-3′) primers. The PCR products were purified and pooled. The pooled samples were then separated using 2% agarose gel electrophoresis. Fragments ranging from 314 to 414 bp (with indices and adaptors) were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Paired-end sequencing (126 bp from both ends) was performed using an Illumina HiSeq 2500 System (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. To evaluate the accuracy of the SLAF libraries, the japonica rice Nipponbare (Oryza sativa L.) (http://rice.plantbiology.msu.edu/ ) was used as a control with the same process of library construction and sequencing.
5
Maize Genotyping Using SLAF-seq
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Young and healthy maize leaves were collected from the two parental lines and 261 RILs at the seedling stage, frozen in liquid nitrogen, and stored at –80 °C. Total genomic DNA was extracted using the cetyltrimethylammonium ammonium bromide (CTAB) method [48 (link)]. All samples with a suitable concentration and quality were used for library construction.
The library was constructed using the SLAF sequencing method described previously [49 (link)]. Two restriction enzymes, Hpy166II and HaeIII (New England Biolabs, Ipswich, Massachusetts, USA), were selected to digest the genomic DNA into 414- to 464-bp fragments. The ends of completely digested fragments were repaired into blunt-ended DNA and the 5' ends were phosphorylated. An adenine base was added to the 3' end of the fragment, after which the duplex index sequencing adapter was connected to the A-tailed fragment. The target fragment was amplified, purified, and sequenced on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, California, USA).
The library was constructed using the SLAF sequencing method described previously [49 (link)]. Two restriction enzymes, Hpy166II and HaeIII (New England Biolabs, Ipswich, Massachusetts, USA), were selected to digest the genomic DNA into 414- to 464-bp fragments. The ends of completely digested fragments were repaired into blunt-ended DNA and the 5' ends were phosphorylated. An adenine base was added to the 3' end of the fragment, after which the duplex index sequencing adapter was connected to the A-tailed fragment. The target fragment was amplified, purified, and sequenced on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, California, USA).
6
Genomic DNA Extraction and SLAF Library Construction for Cyprinidae
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Genomic DNA was extracted from muscle tissue using the standard phenol-chloroform method [23 ]. DNA quality was first assessed using 1% agarose gel and was further quantified using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). SLAF sequence strategy with specific modifications was utilised in library construction. Briefly, the reference sequence of Cyprinus carpio (GenBank accession number: GCA_000951615.2) [24 (link)] was used to conduct the pre-experiment on silico simulation of the number of markers generated by various endonuclease combinations. The SLAF library was constructed based on the SLAF pilot experiment in accordance with the predesigned scheme, and eventually two endonuclease combinations of HaeIII and Hpy166II (New England Biolabs, Ipswich, MA, USA) were applied to genomic DNA digestion in the fish populations. Zhang et al. described the details of the SLAF sequence strategy [25 (link)]. The sequencing data were generated using Illumina HiSeq2500 platform. Raw data had been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive with the Bioproject number PRJNA599030.
7
SLAF-seq for Genetic Mapping
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SLAF-seq method13 (link) was used. First, the genome of the parents and F2 population was digested by Hpy166II (New England Biolabs, MA, USA). Then polyA as dATP was ligated to the end of the digested fragment by employing the Klenow fragment (3′–5′ exo-) (New England Biolabs) at 37 ℃. Next, the PAGE-purified dual-label sequencing markers (Life Technologies, CA, USA) were ligated to the newly added terminal polyA utilizing the T4-DNA ligase. PCR was performed with the diluted DNA samples, and the forward: 5′-AATGATACCGACCACCGA-3′ and reverse: 5′-CAAGCAGAAGACGGCATA-3′ primers, Q5® High-Fidelity DNA Polymerase (NEB), and dNTPs. The PCR products were purified and collected by Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) and separated by electrophoresis on a 2% agar gel. The DNA fragment (with indices and adaptors) between 264 and 464 bp was electrophoresed again, and the band was extracted from the gel employing a QIAquick® gel extraction kit (Qiagen, Hilden, Germany). The paired, terminal 125 bp sequences obtained were analyzed on a Hi-Seq 2500 system (Illumina Inc., CA, USA).
8
Improved SLAF-seq Strategy for Marker Discovery
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In this study, an improved SLAF-seq strategy was used. First, for the in silico prediction of the number of markers produced by different enzymes, marker-discovery experiments were designed by analyzing the ‘WK10039’ radish reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_000801105.2 ). The genomic DNA of the samples included in the SLAF pilot experiment was digested with Hpy166II and HaeIII (New England Biolabs, Beverly, MA, USA). Dual-index sequencing adapters were ligated to the digested fragments using T4 ligase (New England Biolabs). PCR amplifications were performed using appropriate concentrations of the prepared DNA samples. Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) were used to enrich the PCR products, which were 364–464 bp long (with sequencing adapters). Finally, diluted gel-purified products were sequenced using the Illumina HiSeq 2500 system (Illumina, Inc., San Diego, CA, USA) to generate 125-bp paired-end reads. The sequencing was performed by Biomarker Technologies Corporation (Beijing, China).
9
Quantitative LAMP Reaction Analysis
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The MATLAB script processes a .txt file with temperature-time data generated from the TE Tech Controller and a TIF stack containing 2-channel images of the LAMP and melt curve from the LEICA microscope. Partitions are identified using a custom iterative thresholding algorithm and labels are propagated throughout the TIF stack using a custom labeling algorithm. Average well intensity is tracked over time to generate LAMP curves and plotted against temperature to generate the melt curves. Complete details of the script are in the Supplementary Materials and Methods , ‘MATLAB script.’
Bulk LAMP reactions were conducted in 10 μl volumes within a well plate on a CFX96 Real-time Thermocycler (Bio-Rad) at buffer conditions and temperatures matching the dLAMP reactions.
Enzymatic digestions of bulk LAMP products were conducted using CAC8I (#R0579S), Hpy166II (#R0616S), ACCI (#R0161S), AciI (#SR0551S), MseI (#R0525S) and HpyCH4III (#R0618S) purchased from New England Biolabs and were conducted in 50 μl reaction volumes containing 1 μl enzyme, 1 μg DNA, in 1 × Cut Smart Buffer and incubated for 1 h at 37°C. Samples were inactivated for 1 h at 80°C and diluted to 1 ng/μl (∼1:300) to run on an Agilent 4200 TapeStation using High Sensitivity D5000 ScreenTape (#5067-5592) with ladder (#5190-7747), and D100 ScreenTape (#5067-5584) with High Sensitivity D1000 Reagents (#5067-5585).
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