Tag Archives: read length

150 bp, 250 bp and next year 300 bp:
Illumina keeps the competition on the go

Illumina is currently in the midst of the MiSeq sequencer updates. The software update, the new flowcells and the new sequencing chemistry enable runs with outputs of around 8 Gbp and 250 bp read length. The first updates have reached Europe just recently and only a few days ago our own MiSeq has received the update.

That’s not the end of the story for Illumina. Just a week ago, they already have announced the next update. In the second half of 2013 Illumina is planning to offer another MiSeq update that will increase the output to 15 Gbp. They achieve this tremendous output for their benchtop device by increasing the read length to 300 bp and resolving about 25 million clusters on the flowcell.

Considering the intense competition with Life Tech’s Proton and Ion Torrent sequencer, Illumina needs to steadily improve the specs of their sequencing devices. In March, Life Tech plans to increase the output of their Proton sequencer to around 36 Gbp. That’s still a bit more than the new MiSeq upgrade can deliver, but one also has to evaluate the differences in the read length. While the MiSeq will be able to produce 300 bp reads soon thereafter, the Ion Proton is generating reads from 100 to 150 bp. And the difference is even more remarkably when the sequencing on the MiSeq is performed with the paired-end module – an approach that is not possible with Life Techs devices. By using library insert sizes of around 450 – 500 bp, the two overlapping reads can generate a single consensus read of about that size.

In my opinion the Illumina MiSeq is at the forefront of the race and if Illumina’s plan works out they will be there in 2013, too. But we all know how short-lived the NGS market is. So let’s see what’s coming!

Survey Result:
Applications Using Roche GS Technology Providing Read Length <700 bp

We asked, for which kind of application do you use the Roche GS FLX+, the GS FLX Titanium or GS Junior sequencing technology providing read length of up to 700 bp? 36 people answered the poll:

 

 

 

 

 

 

 

 

 

Please find on the right hand side our new poll and add your voice!

The MiSeq Will Further Challenge the Roche 454 FLX+ Technology

Currently, Roche 454 has a unique selling proposition in providing the only sequencing technology on the market delivering long reads with high accuracy at the same time. Just last year Roche 454 launched the new GS FLX+ chemistry delivering reads with a modal read length of up to 700 bp. Long reads are crucial for de novo sequencing of genomes and transcriptomes and for sequencing of amplicons.

The current version of the Illumina MiSeq enables 2x 150 bp paired-end reads and 1.5 – 2 Gbp per run, which is slightly over the output of a typical GS FLX+ run. When working with short insert libraries of 200-250 bp, both paired-end reads will overlap and finally generate one longer read of up to 250 bp.

This read length is still not in a competitive range for Roche 454, but recently Illumina announced the launch of a MiSeq instrument upgrade by the middle of the year. According to Illumina ‘s vice president of marketing, the upgraded instrument will generate 2x 250 bp paired-end reads and up to 7 Gbp of data output. When sequencing short insert libraries with the 2x 250 bp paired-end module, reads with up to 450 bp can be generated. Thus, read length comes again closer to the read length of the Roche FLX+ technology (not to mention the 10x higher data output).

We have to wait and see whether the MiSeq upgrade keeps what Illumina promises, but for me personally it is quite clear, that with the announced specifications, the MiSeq will sooner or later replace Roche 454 sequencing for certain applications. In this light it is very interesting that Roche offered a friendly take over of Illumina this week.

 

Impact of Read Length and Paired End Sequencing on BAC Assembly Quality

 

Photographer: Florian Gerlach from Nawaro

Photographer: Florian Gerlach from Nawaro

Does read length really matter when doing a de novo assembly of BACs from highly repetitive plant genomes, like that of barley? Will paired end sequencing improve the assembly considerably? Is it essential to barcode each BAC clone before sequencing? 

These are the main questions of a study from Taudien et al, published in September 2011, answered by comparing assembly data derived from different sequencing data sets.

To investigate the effect of the read length the authors compared the assembly quality from 4 BAC clones with either FLX or with Titanium chemistry reads at equal sequencing depth. Even though the average read length differed by only 40 bp (223 vs 263 bp) the assemblies with the longer reads were considerably better. They contained fewer missassemblies and reduced number of gaps. According to the authors this is mainly due to the broader read distribution of the Titanium chemistry, where a fraction of reads displayed a read length of >600 bp.

These findings are in accordance with our experience from BAC assemblies. Having performed several BAC sequencing projects with GS FLX+ yet (average read length of 550-650 bp and some reads up to 1000 bp), we can report here that FLX+ read length has the potential to even more improve the BAC assembly quality. 

Please have a look at the research article for further information.

GS FLX+ Sequencing Successfully Established

Being the first service provider in Germany having our GS FLX sequencers updated to GS FLX+ technology, we have meanwhile successfully established the sequencing of shotgun, long paired end (LPE) and cDNA libraries with GS FLX+. The FLX+ business is fully integrated in the daily routine of our production team and we like to share our experiences on run performance with you:

Of course the read length differs in dependence on the organism to be sequenced, but average read length of 600-700 bp has been proven to be a realistic value for sequencing of shotgun libraries so far. Also the announced yield of at least 1 million reads per full run could be confirmed in practice: All shotgun runs performed so far surpassed this specification.

When sequencing a shotgun library, data output per segment therefore almost doubled, making the Roche 454 sequencing more attractive again. Sanger-like read length are of very high value for any de novo sequencing approach whether for a genome or a transcriptome project.

When you are interested in our FLX+ sequencing service we invite you have a look at our current special GS FLX+ offers.