你所不知道的各种seq技术

随着高通量测序技术的普及，各种seq技术层出不穷。前几天有学员问了一个*seq，居然一下子把我问住了，因为我也没听过这个seq。其实各种seq技术核心都是一样的，只不过测序的样品不同。这里我们转载“生信开发者”微信号总结的各种seq概念，以供读者自行查阅和学习。

Whole Genome Sequencing(WGS)

Whole-genome sequencing is the most comprehensive method for analyzing the genome. Genomic information has been instrumental in identifying inherited disorders, characterizing the mutations that drive cancer progression, and tracking disease outbreaks. Rapidly dropping sequencing costs and the ability to produce large volumes of data with today’s sequencers make whole-genome sequencing a powerful tool for genomics research.
While whole-genome sequencing is commonly associated with sequencing human genomes, the scalable, flexible nature of next-generation sequencing (NGS) technology makes it equally useful for sequencing any species, such as agriculturally important livestock, plants, or disease-related microbes.

Whole Exome Sequencing(WES)

Perhaps the most widely used targeted sequencing method is exome sequencing. The exome (the protein-coding region of the human genome) represents less than 2% of the genome, but contains ~85% of known disease-related variants,1 making whole-exome sequencing a cost-effective alternative to whole-genome sequencing.
Exome sequencing can efficiently identify coding variants across a wide range of applications, including population genetics, genetic disease, and cancer studies.

Mate pair sequencing

Mate pair sequencing involves generating long-insert paired-end DNA libraries useful for a number of sequencing applications, including:

De novo sequencing

Genome finishing Structural variant detection Identification of complex genomic rearrangements Combining data generated from mate pair library sequencing with that from short-insert paired-end reads provides a powerful combination of read lengths for maximal sequencing coverage across the genome.

ChIP-Seq

Isolation and sequencing of genomic DNA “bound” by a specific transcription factor, covalently modified histone, or other nuclear protein. This methodology provides genome-wide maps of factor binding.

RNA-Seq

Extraction, fragmentation, and sequencing of RNA populations within a sample. The replacement for gene expression measurements by microarray. There are many variants on this, such as Ribo-Seq (isolation of ribosomes translating RNA), small RNA-Seq (to identify miRNAs), etc.

MicroRNA-seq

MicroRNA sequencing (miRNA-seq), a type of RNA-Seq, is the use of next-generation sequencing or massively parallel high-throughput DNA sequencing to sequence microRNAs, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is often enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs. Evidence that dysregulated miRNAs play a role in diseases such as cancer has positioned miRNA-seq to potentially become an important tool in the future for diagnostics and prognostics as costs continue to decrease. Like other miRNA profiling technologies, miRNA-Seq has both advantages (sequence-independence, coverage) and disadvantages (high cost, infrastructure requirements, run length, and potential artifacts).

CLIP-seq

CLIP (cross-linking immunoprecipitation) is a method used in molecular biology that combines UV cross-linking with immunoprecipitation in order to analyse protein interactions with RNA or to precisely locate RNA modifications (e.g. m6A). CLIP-based techniques can be used to map RNA binding protein binding sites or RNA modification sites of interest on a genome-wide scale, thereby increasing the understanding of post-transcriptional regulatory networks.

PAS-seq

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs.

GRO-Seq

RNA-Seq of nascent RNA. Transcription is halted, nuclei are isolated, labeled nucleotides are added back, and transcription briefly restarted resulting in labeled RNA molecules. These newly created, nascent RNAs are isolated and sequenced to reveal “rates of transcription” as opposed to the total number of stable transcripts measured by normal RNA-seq.

5'RNA-Seq/5'GRO-Seq

Sequencing only the 5' cap-protected fragments of RNA can be used to define sites of transcriptional initiation at nucleotide resolution. This section covers the identification of TSS from 5' RNA sequencing data.

Hi-C-seq

Genomic interaction assay for understanding genome 3D structure. This assay is much more specialized - For more information about how to use HOMER to analyze Hi-C data, check out the Hi-C analysis section.

DNase-Seq

Treatment of nuclei with a restriction enzyme such as DNase I will result in cleavage of DNA at accessible regions. Isolation of these regions and their detection by sequencing allows the creation of DNase hypersensitivity maps, providing information about which regulatory elements are accessible in the genome.

BS-Seq/methyC-Seq

Profiling of cytosine methylation in genomic DNA.

Drop-seq

Drop-seq is a technology we developed to enable biologists to analyze RNA expression genome-wide in thousands of individual cells at once. We first described this in a 2015 paper in Cell. Though commercial implementations of droplet-based single-cell RNA-seq also now exist, we have made Drop-seq open-source and want to make sure that any lab can build their own system. The materials for constructing a Drop-seq setup in one’s own lab can be obtained for about $6,000. The reagents for performing Drop-seq cost about 6 cents per cell.

RAD-seq

Restriction-site associated DNA sequencing, a method that samples at reduced complexity across target genomes, promises to deliver high resolution population genomic data—thousands of sequenced markers across many individuals—for any organism at reasonable costs. It has found application in wild populations and non-traditional study species, and promises to become an important technology for ecological population genomics.

CAPP-Seq

CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) is a sensitive method used to quantify DNA in cancer. It measures Cell-free tumor DNA which is released from dead tumor cells into the blood. This method can be generalized for any cancer type that is known to have recurrent mutations.[1] CAPP-Seq can detect one molecule of mutant DNA in 10,000 molecules of healthy DNA.
CAPP-Seq was designed to lower sequencing costs by only targeting specific areas of the genome that are recurrently mutated for a given cancer. This allows for sequencing costs between 200−300 USD. It can also target multiple areas of the genome at once and a variety of different types of mutations, allowing for a lower amount of input DNA compared to other methods.