Tag Archives: Amplicon sequencing

Amplicon Sequencing Strategy: What Is Your Technology Of Choice?

amplicon_sequencingWe asked for your favourite technology for amplicon sequencing.

Please find the results here:

  • The majority (41 people) voted for Illumina MiSeq due to the data output
  • 29 prefer Ion Torrent for amplicon sequencing
  • 24 favour Roche 454 because of the long reads
  • 6 people say that classical Sanger sequencing is their technology of choice
  • Just 4 are using other technologies

104 NGS experts took part in the voting.

 

800 bp Read Length For Amplicon Sequencing Is Not Science Fiction

Amplicon sequencing with Roche GS JuniorAbout a year ago my colleguage Regina reported about the new possibilities of using the MiSeq system for amplicon sequencing (16S Amplicon Experiments: Which Platform to Choose?). Now, one year later still everything is true about the advantages of amplicon sequencing using the MiSeq (e.g. lower cost/base).

The main advantage of the Roche system are the long reads that are highly valuable for some applications. By ligating appropriate sequencing adaptors we can currently deliver average read length of up to 700 bp when using the GS FLX+ pipeline. Further improvements regarding the read length can be expected with the launch of a new amplicon pipeline from Roche for the Roche GS FLX+ system (planned for summer 2013).

And beside the ultra long reads on the GS FLX+ system there are still some advantages of amplicon sequencing using the GS Junior system compared to other technologies:

+ short turnaround time (starting from 5-10 working days)

+ competitive pricing

+ moderate to long reads (350 – 450 bp)

+ sufficient data output for all projects with a medium size of samples (e.g. up to 24)

What is your preferred next generation sequencing technology for amplicon sequencing? Take part in our current poll.

Spring Special – Amplicon Sequencing


  • Would you like to detect variances down to a frequency of 0.1% in your PCR sample?
  • Are you interested in a short turn around time?
  • You need primers for your amplicons?

 

Amplicon sequencing is still under discussion. Which technology is most suitable? In one of our latest blog posts we discussed this issue as well. For this years spring special we therefore decided to create a new NGS Favourite – a one stop solution for Amplicon sequencing. By sequencing your amplicons on the GS Junior we will be able to deliver the data in a short turnaround time while you will still profit from the long reads the FLX chemistry provides. Furthermore you will get comprehensive bioinformatic data that will be able to answer already questions like: how many clusters were obtained or what is the homology of each read compared to the representative read. This data will help you for example to analyse metagenomes in your environmental samples like soil, water or gut.

Additionally we as a service provider for oligonucleotide synthesis, gene synthesis, custom DNA sequencing and NGS are able to offer you primers for free for your amplicon sequencing approach if you order our Spring Special.

Contact us if you are interested in a spring special quote or read more on our website. This offer is valid until 30.06.2012.

Is 16S rRNA Sequencing Ready for Illumina Sequencer?

Claesson et al (2010) compared for the first time 16S rRNA metagenome sequencing on Roche GS FLX and Illumina GAIIx. Several tandem combinations of variable regions were targeted in microbial DNA extracted from fecal sample material and sequencing of the PCR amplicons was performed in a quarter segment of a full GS FLX run and a full lane of GAIIx with 2x 100 bp module.

Comparison revealed that taxonomic classification down to genus level is a lot worse for Illumina reads because of their shorter read length and higher error rate. Also the insight into rarely occurring species is far from increasing considerably in comparison to GS FLX sequencing, as it might have been expected because of the significantly higher coverage.

As the authors suggest a big step forward to increase classification efficiencies with Illumina technology is sequencing of fragments with insert sizes smaller than 200 bp. Following this strategy forward and reverse read which display poor quality at the read ends are overlapping and thus generate a consensus sequence with improved quality.

For sure respective studies are underway and we will continue to monitor the latest developments concerning this topic.