Tag Archives: paired end library

A Method to Increase Accuracy in Next Generation Sequencing

B) Duplex Sequencing workflow. Sheared, T-tailed double-stranded DNA is ligated to A-tailed adapters. Because every adapter contains<br />a Duplex Tag on each end, every DNA fragment becomes labeled with two distinct tag sequences (arbitrarily designated α and β in the single fragment shown).<br />PCR amplificationwith primers containing Illumina flow-cell–compatible tails is carried out to generate families of PCR duplicates. Two types of PCR products are<br />produced from each DNA fragment. Those derived from one strand will have the α tag sequence adjacent to flow cell sequence 1 and the β tag sequence<br />adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally.

Next-generation sequencing allows detection of minor variants in a heterogeneous sample. However, errors in PCR and sequencing pose limits on its sensitivity.
A group at University of Washington developed a method, called Duplex Sequencing, to dramatically improve accuracy by sequencing both strands of each DNA duplex. Mutations that are detected in the consensus sequence of one strand but not the other are discounted as technical errors.

The authors adopted the method to Illumina sequencing. It involves the use of modified adaptors that have a tag with random sequence attached. After ligation of these modified adaptors, each duplex DNA fragment is flanked by two different tags and subjected to paired-end sequencing. Sequences of the same duplex from the complementary strands can therefore be uniquely identified by having the same tags on either ends. Comparing sequences of the two strands allows identification of true mutations. The authors estimated that Duplex sequencing has a theoretical background error rate of less than one per 109 nucleotides sequenced.
Full text article can be accessed here: http://www.pnas.org/content/early/2012/07/31/1208715109.full.pdf

 

NGS Favourites – Launch

Dear Blog readers,
today I am really delighted to announce the launch of the NGS Favourites.

NGS Favourites are the straightforward solution for your Next Generation Sequencing project. They are based on the wealth of knowledge that we have accumulated from over 5 years of servicing the NGS community and represent optimised packages for common NGS applications.

The NGS Favourites stand out due to:

  • Project-oriented solutions
  • Economic costs
  • Easy ordering

The NGS Favourites are available for different fields of applications:

  • Genome Sequencing Favourites – using shotgun (SG) libraries only or a combination of SG and LPE libraries
  • Transcriptome Sequencing Favourites – receive comprehensive data you can really build on
  • Exome Sequencing Favourites – sequence 6 human Exomes with the Illumina TruSeq Kit
  • Library Service Favourites – get your libraries for GS FLX sequencing prepared from us

Find your suitable Favourite and explore the easy way of sequencing with us as your professional project partner.

Why Choose LJD Libraries Rather Than Mate-Pair Libraries?

Why should you choose long jumping- distance (LJD) libraries rather than mate-pair libraries, especially for de novo sequencing projects? There is a simple answer to this question: Because the resulting data are much more suitable for de novo assembly and scaffolding.

Why is this so? Mate-pair libraries contain higher percentage of undesired inward-facing read-pairs (if you are not familiar with inward and outward facing reads, just take a look into our FAQs on this subject). These reads are not mate-pair (in other words they are shotgun paired-end). The portion of such reads in the LJD library is greatly reduced.

Furthermore, if using mate pair libraries, it is not clear, if (and if yes, where) there is the changeover within the resulting reads. In other words: A read may contain sequence from one AND the other end without knowing where the changeover is. As a result chimeric reads go into the assembly. This effect is almost completely eliminated if LJD libraries are used, because of the differences in library generation.

Which experiences did you gain with LJD or mate-pair libraries? I’d be happy to hear from you.

What Strategy and how much Coverage is Needed for Bacterial de novo Sequencing?

In the NGS (Next Generation Sequencing) group of the networking platform LinkedIn I came across a lively discussion about the best strategy for de novo sequencing of a bacterial genome. The discussion was about technology (Roche versus Illumina), coverage (from 10-fold on Roche GS FLX to 500-fold on Illumina HiSeq 2000) and library types. The comments and advices given coincide with our experience that this question can not be answered without any further information on the genome to be sequenced. GC content, amount and size of repeat structures as well as the genome size of the bacterium have to be considered. We have meanwhile de novo sequenced more than 100 microbial genomes and according to our experience GS FLX technology with a combination of shotgun and long paired end libraries will deliver a high quality genome sequence that is suitable for gap closing projects.

The multiple library approach is described in detail in our Application Note and in a Press Release. Depending on the complexity and size of the genome, we select the appropriate library.