Next generation sequencing (NGS) applied sciences present a high-throughput means to generate great amount of sequence information.
However, quality control (QC) of sequence information generated from these applied sciences is extraordinarily necessary for significant downstream evaluation. Further, extremely environment friendly and quick processing instruments are required to deal with the big quantity of datasets.
Here, we’ve developed an software, NGS QC Toolkit, for quality examine and filtering of high-quality information. This toolkit is a standalone and open supply software freely out there at http://www.nipgr.res.in/ngsqctoolkit.html.
All the instruments within the software have been applied in Perl programming language. The toolkit is comprised of user-friendly instruments for QC of sequencing information generated utilizing Roche 454 and Illumina platforms, and extra instruments to assist QC (sequence format converter and trimming instruments) and evaluation (statistics instruments).
A spread of choices have been supplied to facilitate the QC at user-defined parameters. The toolkit is predicted to be very helpful for the QC of NGS information to facilitate higher downstream evaluation.
Preparation of decreased illustration bisulfite sequencing libraries for genome-scale DNA methylation profiling.
Genome-wide mapping of 5-methylcytosine is of broad curiosity to many fields of biology and medication. A spread of strategies have been developed, and several other have not too long ago been superior to genome-wide scale using arrays and next-generation sequencing approaches.
We have beforehand reported decreased illustration bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich elements of the genome, thereby reducing the quantity of sequencing required whereas capturing the bulk of promoters and different related genomic areas.
The strategy offers single-nucleotide decision, is very delicate and offers quantitative DNA methylation measurements. This protocol ought to allow any normal molecular biology laboratory to generate RRBS libraries of excessive quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate brief fragments that include CpG dinucleotides on the ends.
After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are dimension chosen, subjected to bisulfite conversion, PCR amplified and finish sequenced on an Illumina Genome Analyzer. Note that alignment and evaluation of RRBS sequencing reads usually are not coated on this protocol.
The extraordinarily low enter necessities (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the method’s single-nucleotide decision extends RRBS to a extensive range of organic and scientific samples and analysis purposes. The whole course of of RRBS library building takes ∼9 d.