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Old 03-31-2011, 06:45 AM   #1
Bioo Scientific
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Default The NEXTflex™ PCR-Free DNA Sequencing Kit Reduces Bias in NGS Library Prep

Bioo Scientific announced today that it has launched the NEXTflex™ PCR-Free DNA Sequencing Kit, the first commercial kit that completely eliminates the need for amplification during library prep.

Amplifying AT or GC rich genomic regions often leads to sequence biased nucleotide compositions and poses a serious challenge during analysis. Using specially designed master-mixed enzymes, the NEXTflex™ PCR-Free DNA Sequencing kit completely eliminates the need for amplification, enabling better read mapping and a reduction in duplicate sequences leading to reduced sequence cost, more representative base identities and better de novo assembly. In assessing bias, the presence of low quality and high GC reads often cannot be aligned well against a reference genome. High GC containing profiles tend to shift toward higher GC content, an indication of poor base representation. Eliminating PCR removes this content bias for more representative reads. PCR is also the cause for a significant amount of gene duplicates which increases sequence cost, adapter dimers and noise in cluster detection. According to Dr. Toloue, Bioo Scientific’s Director of Genomic Research, the key to eliminating PCR was developing optimized enzymes tailored specifically for each step of library prep. "Typical off-the-shelf enzymes do not require modifications if you're going to end up amplifying your product at the end. The real concern is after sequencing when you realize that your reads have degrees of bias or are contaminated with duplicates and dimers. In the new NEXTflex PCR-Free DNA Sequencing Kit we have made specialized improvements to each enzyme at every step in the library prep protocol. “

The NEXTflex™ PCR-Free DNA Sequencing Kit features “Enhanced Adapter Ligation Technology” which results in library preps with a larger number of unique sequencing reads. By making improvements to the ligation enzymatic mix, the user will now have the ability to perform ligations with longer adapters and expect to see better binding efficiencies. The NEXTflex™ PCR-Free DNA Sequencing Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. In addition, the availability of up to 48 unique adapter barcodes makes this the most high-throughput kit available. This kit can be used to prepare genomic DNA libraries for sequencing using Illumina®/Solexa® GAII and HiSeq 2000/1000 platforms.


About Bioo Scientific Corporation
Bioo Scientific Corporation is an Austin, TX based biotechnology company that provides innovative solutions to the life science industry. Bioo Scientific offers a complete portfolio of products that increase sensitivity, flexibility and speed to next-generation sequencing.

Bioo Scientific’s products are used around the world and can be purchased both directly or through our global distribution network with markets in more than 40 countries. For more information about Bioo Scientific’s NEXTflex™ DNAseq library preparation product line, visit http://www.biooscientific.com/Produc...e/DNA-Seq.aspx.

Last edited by Bioo Scientific; 06-03-2014 at 11:03 AM.
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Old 04-03-2011, 06:14 PM   #2
link1
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Default barcodes with PCR free prep

We are very interested in amplification free library prep. We are currently using your Nextflex library prep kit. Can we still use all 48 Nextflex barcodes with the the new amplification free library prep ?

thanks
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Old 04-05-2011, 08:12 AM   #3
Bioo Scientific
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Default Barcodes

Yes the NEXTflex™ DNA Barcodes can be used with either the NEXTflex™ PCR-Free DNA Sequencing Kit and the NEXTflex™ DNA Sequencing kit. They are also compatible with mRNAseq applications.
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Old 04-06-2011, 01:08 AM   #4
koadman
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Default Starting material requirements?

Hi! Congrats on the PCR-free product launch. The description on the web site mentions 3ug starting material yielding enough ligation product to load 600 lanes. How much starting material does the protocol require to load just 1 lane? 3ug / 600 = 5ng, but surely a kit user must need more than 5ng starting?
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Old 04-06-2011, 09:48 AM   #5
Bioo Scientific
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Thanks Koadman.

Using the NEXTflex™ PCR-Free Sequencing kit, we recommend starting with 1-3 µg of material. While there might be some variation depending on your starting sample type, 1-3 ug is enough to achieve the 600,000-800,000 clusters /mm2 for the GAIIx or 300,000-600,000 clusters / mm2 for HiSeq. Using qPCR you can quantitate the pM concentrations that you need to start.

The regular NEXTflex™ DNA Sequencing kit (which includes the PCR step) only requires 750 ng - 2.5 µg of starting material for libraries.
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Old 04-07-2011, 04:59 AM   #6
link1
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Does the PCR Free kit use the same adapter DNA ligation technology as the regular NEXTflex kit ? We have been pleased with the ligation in that kit.
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Old 04-07-2011, 01:27 PM   #7
Bioo Scientific
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Hi Link1,

Yes, the NEXTflex PCR-Free DNA Sequencing Kit does use “Enhanced Adapter Ligation Technology” which results in library preps with a larger number of unique sequencing reads. These improvements to the ligation enzymatic mix, allow for ligations with longer adapters and higher binding efficiencies, resulting in a greater number of unique sequence reads.
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Old 04-20-2011, 10:31 AM   #8
luc
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Default NextFlex and DSN treatment

Hi,
Are the "long" adapters of the NextFlex system working well with DSN repeat reduction protocols?
At which point of the library prep would you suggest to do the DSN treatment?

Thanks in advance!
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Old 04-20-2011, 12:40 PM   #9
Bioo Scientific
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The NEXTflex™ DNA Barcodes are compatible with DSN to normalize RNA samples. We recommend first preparing your library, then performing the DSN treatment, clean-up, followed by amplification. The NEXTflex Barcode kits include the PCR primers needed for sample prep and post DSN treatment.
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Old 04-20-2011, 12:46 PM   #10
luc
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Thanks for the quick information !
Quote:
Originally Posted by Bioo Scientific View Post
The NEXTflex™ DNA Barcodes are compatible with DSN to normalize RNA samples. We recommend first preparing your library, then performing the DSN treatment, clean-up, followed by amplification. The NEXTflex Barcode kits include the PCR primers needed for sample prep and post DSN treatment.
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Old 12-20-2012, 07:10 AM   #11
Bioo Scientific
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Default Reduced starting material requirements

A completely gel-free protocol has been incorporated into the NEXTflex™ PCR-Free DNA Sequencing Kit which allows the kit to be used with lower amounts of starting material. The range of DNA that can be used with this kit is now 500 ng – 3 µg.
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amplification free, automation, dna indexed adapters, dna library preparation, pcr free

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