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Next generation sequencing (NGS) Library preparation methods and their biases #
Since 1977, Biomolecules (DNA and RNA) sequencing (Next and Next Next generation) is unrelenting every year and ahead towards personalized medicine (23andme). In the field of sequencing, the key players are in market are Illumina, GS454, Ion Torrent, and SOLiD. Simultaneously, computational biologist around the world developing methods to mining the knowledge from high throughput data and another side molecular biologist optimizes the sample preparation methodologies. These were categories in to upstream and downstream process with respect to sequencers (Figure 1) and the quality of the project is depends on each step in sequential order. Application of NGS are rapidly increasing frequently and the available technologies (Library preparation, Sequencer and Computational tools) also optional according to design of projects and cost affordability. In every state of methodologies there will be inherited biases. A detailed knowledge of these biases will be essential for a careful interpretation of NGS data and it will help to improve library quality or to develop bioinformatics tools to compensate the biases. Here I tried to summarize those steps more briefly.
Sequencing Projects #
For every application of DNA/RNA sequencing, such as metagenomics, transcriptome sequencing, genome sequencing or re-sequencing, chromatin immuno-precipitation sequencing (ChIP-Seq) and so on, there is the need for a specific protocol to convert the source material into a standard DNA/RNA library suitable for loading onto the sequencing instrument. Such as below
DNA Sequencing #
To convert DNA into a sequenceable library, the source DNA must be include the below steps
1) Fragmention (physical or enzymatic methods),
2) Adapter ligation (Double stranded adapters (FLX and SOLiD), Y shaped adapter (Illumina)),
3) Size selection (agarose gel electrophoresis and solid-phase reversible immobilization (SPRI)),
4) Amplification (emulsion PCR (FLX, SOLiD), Surface PCR (Illumina)),
5) Purification and quantification (Mass to molar concentration)
RNA Sequencing (ribosomal and non-ribosomal) #
Library preparation for eukaryotic transcriptomes, polyadenylated mRNAs are commonly extracted using oligo-dT beads, or alternatively rRNAs can be selectively depleted and for prokaryotes, as their mRNAs are not polyadenylated, So RNA is often fragmented to generate reads that cover the entire length of the transcripts which explained in references [1, 2]. Most of the procedures were same as DNA and RNA sequencing includes cDNA making protocols.
Single Molecule RNA preparations #
DNA Biasis #
RNA Biasis #
Type of sequencers: #
454 (emulsion PCR) (http://www.454.com/) #
Illumina (surface PCR) (http://www.illumina.com/) #
- Error Rate <0.1%, Type of errors is Substitution
SOLiD (emulsion PCR) (http://www.appliedbiosystems.com/) #
Other technologies for single molecule real time sequencing #
Pacific Biosciences PacBio RS II system (SMRT)
Linnarsson S: Recent advances in DNA sequencing methods - general principles of sample preparation. Experimental cell research 2010, 316(8):1339-1343.
van Dijk EL, Jaszczyszyn Y, Thermes C: Library preparation methods for next-generation sequencing: tone down the bias. Experimental cell research 2014, 322(1):12-20.
Head SR, Komori HK, LaMere SA, Whisenant T, Van Nieuwerburgh F, Salomon DR, Ordoukhanian P: Library construction for next-generation sequencing: overviews and challenges. BioTechniques 2014, 56(2):61-64, 66, 68, passim.
Detail Lectures #
Sequencing animation (http://www.youtube.com/watch?v=_ApDinCBt8g)
Sequencing Technology explanation (http://www.youtube.com/watch?v=PMIF6zUeKko)