CRM007 Chromogenic E. coli O157

The NGS Single Stranded DNA Library Prep Kit (illumina platform) was developed for construction of high quality libraries using sheared single stranded DNA (50 ng – 500 ng) as input. The kit is ideal for making NGS libraries with samples of denatured DNA, viral DNA, highly degraded ancient DNA and other single stranded DNA. The workflow of the ssDNA kit is simple: make the libraries in two steps followed by PCR and cleanup steps. The libraries will be ready in just 1.5 hours with a 10 minutes of hands-on time. The library pooling is possible dependent on the type of index. The final library is strand specific.

NGS Single Stranded DNA Library Prep Kit workflow

Three index types are available for the kit:

Non-index (Cat.# 30081): Libraries do not have index.

Index(Cat.# 30082): Each of the index primers has a unique 6-base index sequence that can be used to identify libraries. ssDNA library multiplexing is up to 48 samples. Index information can be downloaded here.

Unique dual index (Cat.# 30083): ssDNA sample multiplexing up to 96 libraries is possible with unique dual indexes. We have developed a Four-Base Difference Index System. This makes it possible to generate indexes with at least 4 bases different from each other in the 8 bases index length. The unique dual indexing primers remove NGS errors (example: de-multiplexing errors, read mis-assignment, index hopping etc). Index information can be downloaded here.

Kit advantages:

• • • Quick and simple Protocol
      • Quick: Total protocol time is only 1.5 hours
      • Simple: Hands-on time only 10 minutes
    • Easy procedure based on:
      • The ready-to-use master mix: Made reaction setup easy
      • Less reaction components: Simplify the reaction preparation
    • Less magnetic beads needed: Reduced more than 50% of the beads for cleanup steps
    • Directional library
    • Single stranded DNA as input: From 50 ng to 500 ng

NGS Single Stranded DNA Library Prep Kit has similar library conversion efficiency and yield as compared to a regular DNA library prep kit.

Alignment rate and duplication rate: comparison of single stranded DNA library kit versus double stranded DNA library prep kit.

The NGS Single Stranded DNA Library Prep Kit (illumina platform) was developed for construction of high quality libraries using sheared single stranded DNA (50 ng – 500 ng) as input. The kit is ideal for making NGS libraries with samples of denatured DNA, viral DNA, highly degraded ancient DNA and other single stranded DNA. The workflow of the ssDNA kit is simple: make the libraries in two steps followed by PCR and cleanup steps. The libraries will be ready in just 1.5 hours with a 10 minutes of hands-on time. The library pooling is possible dependent on the type of index. The final library is strand specific.

The series of DNA Size Selection Kits (Magnetic Beads) were developed for DNA size selection using magnetic beads. A total of 11 kits are available, with different selection ranges spanning from 50 bp to over 10 kb. The kits provide a simple and quick approach for the enrichment of a specific range of DNA fragments. The kit workflow allows double-sided or single-sided size selection for specific size cutoffs.

Gel images of different ranges of size selection. Sheared human genomic DNA was used as input.

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DNA size selection is a selective capture of DNA fragments of a specific range of size for next-generation sequencing (NGS) library preparations, PCR, ChIP assay, DNA ligations, endonuclease digestions, adapter removal, and other genomics and molecular biology applications. DNA size selection is preferred after NGS library prep in most of the cases. The NGS library preparation is related to the quality of the sequencing data. Precise NGS library size selection can increase sequencing efficiency, improve data quality, and reduce costs.

There are two types of sequencing technologies: short-read sequencing and long-read sequencing. Short-read sequencing uses DNA libraries that contain small insert DNA fragments of similar sizes, usually several hundred base pairs. The sequencing efficiency can be improved if the DNA size selection is in the right range. Cat.# 20104S and 20104L are the best kits for NGS library size selection of illumina paired-end 100 (PE100) sequencing with 100-200 bp library inserts; Cat.# 20105S and 20105L are the best kits for NGS library size selection of illumina paired-end 150 (PE150) sequencing with 150-300 bp library inserts; and Cat.# 20106S and 20106L are the best kits for NGS library size selection of illumina paired-end 300 (PE300) sequencing with 300-600 bp library inserts.

Long-read sequencing uses a large DNA fragment as input and makes very long reads. Usually, library size selection is preferred to remove smaller fragments. Cat.# 20110S and 20110L are the best kits for long-read sequencing size selection with DNA sizes >5 kb, and Cat.# 20111S and 20111L are the best kits for long-read sequencing size selection with DNA sizes >10 kb.

The magnetic beads technology uses paramagnetic particles, also known as SPRI (Solid Phase Reversible Immobilization) beads, to bind DNA reversibly and selectively. DNA fragments can be size-selected and purified by changing the properties of the magnetic beads or SPRI beads. The magnetic beads can easily separate the beads-binding DNA from the contaminants and unwanted components in the samples. The samples after DNA size selection are free of contaminants such as buffer components, enzymes, proteins, salts, dNTPs, primers, and adapters. Our proprietary magnetic beads reagents improve yield, selectivity, and reproducibility.

Specific DNA fragments at a certain length range can be purified simply using magnetic separation with different beads components, avoiding tedious and time-consuming gel extraction and column-based purification. The magnetic beads method is popular for common DNA size selection, including library size selection. The first beads-binding step, referred to as the right-side clean-up, removes large DNA fragments. The large DNA fragments are bound to the beads and are discarded. The desired DNA fragments in the supernatant are transferred to a new well, and new beads are added to the supernatant for the second beads-binding, referred to as the left-side clean-up. The double-size selected DNA fragments are eluted after ethanol rinsing.

DNA size selection with dual clean-ups.

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A single clean-up is needed for DNA size selection with large fragments. In this case, only the large DNA fragments are bound to the beads. The selected larger DNA fragments are eluted after ethanol rinsing.

DNA size selection with single clean-up for >5 kb and >10 kb DNA.

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Features of DNA size selection and library size selection

• • • High specificity and high recovery of size selection
    • 11 selection ranges are available, including 5 ranges for NGS library size selection
      • • 50-100 bp
        • 100-200 bp
        • 200-500 bp
        • 250-350 bp: ideal for illumina PE100 sequencing
        • 300-450 bp: ideal for illumina PE150 sequencing
        • 450-750 bp: ideal for illumina PE300 sequencing
        • 500-1000 bp
        • 1-3 kb
        • 1-5 kb
        • >5 kb: ideal for long-read sequencing
        • >10 kb: ideal for long-read sequencing
    • Fast and simple
      • • 20-min protocol
        • No gel purification required
        • No columns required
        • No centrifugation required
    • Efficient removal of contaminants and unwanted components