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**GenoMax** · 07-16-2013, 03:27 AM

Originally posted by rjames View Post

Hi!
I am a novice in this field. I just started. I will be working on Ion Torrent PGM within a month. I have a very basic problem. What are single end reads and why are they not preferred for genome assembly? Is there any suite for checking the quality of the sample in NGS? Any site or materials where i can get extensive details about the above questions?

Thanks.
RJames

Most NGS protocols require you to fragment the original DNA molecule so that you end up with fragments that are 350 bp -a few kb in size. If you only sequence one end of these resulting fragments then you do not get spatial information for the entire fragment (since you know the size of the fragment and the length of bases sequenced from both ends in a paired-end sequencing experiment) with just single-end reads. Paired end reads are also useful for locating spliced exons and/or translocations/breaks etc. They can help with unique placement of reads specially if one of the ends happens to be a repetitive sequence.

Every sequencing technology has inherent biases/errors. If you are only planning to use Ion Torrent data and are planning to assemble a de novo genome/transcriptome then you should take these limitations into consideration as you plan your experiment.

Shining a Light on Dark Sequencing: Characterising Errors in Ion Torrent PGM Data

http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1003031

Author Summary DNA sequencing is used routinely within biology to reveal the genetic information of living organisms. In recent years, technological advances have led to the availability of high-throughput, low-cost DNA sequencing machines (‘sequencers’). In 2011, Life Sciences released a new sequencer, the Ion Torrent Personal Genome Machine (PGM). This is the first sequencer to measure changes in pH rather that emitted light to register sequencing reactions. Consequently, this unique technology is both cost-effective and advertised to have high accuracy, making it attractive for many laboratories. However, every sequencing technology introduces unique errors and biases into the resulting DNA sequences, and understanding PGM-specific characteristics is crucial to determining suitable applications for this new technology. We comprehensively examine the types of errors and biases in PGM-sequenced data across several experimental variables, including chip density, template kit, template DNA and across two machines. Using statistical approaches, we quantify the influence of experimental variables, as well as DNA sequence-specific effects, and find that the PGM has two types of technology-specific errors. We also find that the accuracy of the PGM is poorer than that of light-based technologies, and we make recommendations for this technology as well as provide statistical models for overcoming PGM sequencing errors.

Application Unavailable | Springer Nature

http://genomebiology.com/2013/14/5/R51/abstract

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http://www.ncbi.nlm.nih.gov/pubmed/22522955

Some links for assembly:

Next Generation Sequencing (NGS)/De novo assembly - Wikibooks, open books for an open world

http://en.wikibooks.org/wiki/Next_Generation_Sequencing_%28NGS%29/De_novo_assembly

Next Generation Sequencing (NGS)/De novo RNA assembly - Wikibooks, open books for an open world

http://en.wikibooks.org/wiki/Next_Generation_Sequencing_%28NGS%29/De_novo_RNA_assembly

There are several QC packages available for NGS data (FASTQC is one of the most popular) http://seqanswers.com/wiki/Special:B...uality_Control

**rjames** · 07-16-2013, 05:14 AM

Thanks.

Thanks a lot. Those articles are really helpful. They helped immensly.

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