In the past four years, high throughput DNA sequencing platforms have become widely available, leading to nose-diving in the cost of DNA sequencing. The speed and accuracy of the platforms has led to wide acceptance and empowerment among individual investigators. In fact the progress in parallel fields such as data storage and computational skills have supported the growth in next generation sequencing technology.

Next-generation DNA sequencing has changed the way genomic research is done today, by enabling comprehensive analysis of genomes, transcriptomes and epigenome without significant effort. Genetic variation, protein-DNA interaction, noncoding RNA expression profiling etc. can be assessed by Next-generation sequencing. The next gen sequencing or short read sequencing applications could range from identifying etiology and new variations that lead to increased risk of several chronic disorders and diseases, enhanced characterization of livestock genome sequence, identification of virulence markers that can cause diseases in crops and inference of population structure in microbial ecology studies. Major Next-Gen Sequencing platforms today are Roche 454, Illumina Genome Analyzer and ABI SOLiD™.

 

Platforms	Roche (454) GS-FLX	Illumina Genome Analyzer II system	ABI SOLiD
Starting DNA (μg)	3 – 5	0.1 – 1	0.1 – 20
Amplification	Emulsion PCR	Bridge PCR	Emulsion PCR
Sequencing method	Pyrosequencing	Sequencing by synthesis	Sequencing by ligation
Read length (bases)	250	32-40	35
Throughput capability (Gb per run)	0.1	1.3	4
Reagent cost per run (list prices)	8500	3000	3400
Run time	7.5 h	3 d	7 d
Ref: Applications and Case Studies of the Next-Generation Sequencing Technologies in Food, Nutrition and Agriculture. George E. Liu*. Recent Patents on Food, Nutrition & Agriculture, 2009, 1, 75-79

 

Next Generation Data Analysis deliverables

Whole Genome & targeted re-sequencing	Transcriptome analysis	Small RNA analysis	De novo assembly	ChIP-Seq analysis
SNVs	Read counts	Reads counts	Asembled contigs and scaffolds	Protein bound regions
Small insertions and deletions	RPKM values	Mapped reads in BAM format	Quality reports	Motif analysis
Structural variations	Expressed SNVs	Coverage files (wig format)		Mapped reads in BAM format
Mapped reads in BAM format	Alternative splice events	Quality reports		Coverage files (wig format)
Coverage files (wig format)	Novel transcribed regions			Quality reports
Quality reports	Mapped reads in BAM format
	Coverage files (wig format)
	Quality reports

 

QA/QC reports

Primary analysis	Secondary analysis	Tertiary analysis
Summary on reference sequence or genomic reference (length, composition, gaps)	Summary of matching results	SNP frequency report
Reports on raw data based on quality values a. Avg. quality value by base position of the read b. Distribution of quality values	Coverage distribution (for each chromosome/reference sequence)	Report on indel sizes
Summary of raw read sequences	Regions not covered after mapping	Distribution of expression values (ex. RPKM values)
Total number of reads by tile/panel	Distribution of matched read lengths	Distribution lengths of peak regions
Base composition by base position of the read	Error rates by base position of the read sequence	Distribution of small RNA lengths
Distribution of read lengths		Data visualization
		De novo assembly report (N50 calculation, distribution contig lengths, base compostion of assembled contigs), and etc…

 

Some techniques in Next Generation Genomics that have transformed Genomics research are as follows:

The need for identifying causative mutations, low frequency SNP’s and genome variation within populations is the fundamental objective of targeted and whole genome resequencing. This requires analysis of millions of sequences and that the read length is sufficiently longer to be mapped onto the genome accurately. In addition the mapping process itself must be accurate and quick. The next generation sequencing platforms have a clear edge over the electrophoresis based sequencing in such cases. These systems generate gigabytes of data in a single run providing full genome coverage.

De novo sequencing allows generation of primary genetic sequence of an organism. With longer read lengths and faster data analysis, de novo sequencing is much cheaper and quicker compared to Sanger sequencing.

Small RNA can serve as important biomarkers for diagnostic purposes. Digital gene expression using next generation genomic technologies can help in discovery and analysis of RNA without the need for previous sequence information. These systems are highly accurate and suited for analyzing low RNA expression levels.

ChIP-Seq data sets analysis and annotation for identification of protein-DNA interactions of an entire genome.

Metagenomics annotations and classification requires high throughput or short read sequencing technologies in cases where DNA is purified from an environmental sample and sequenced. The sequences could be from several different species that need to be assembled.