Next Generation Sequencing

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Committed to Sequencing Excellence.

Macrogen provides wide range of sequencing and bioinformatics services to academic, pharmaceutical and clinical research institutions around the world. Established in 1997, Macrogen has been a reliable partner for more than 18,000 customers in 153 countries. We provide end-to-end genetic analysis services suitable for publication. By using verified products of accredited vendors and manufacturers, we assure high quality results with consistency and repeatability. At Macrogen, every sequence count.

What is Next Generation Sequencing?

Today, Next Generation Sequencing (NGS) technology has made it possible to process and decode the entire human genome in as little as few days;, revolutionizing the approach to study the genome. NGS technology applications enable unprecedented scalability and touch on all areas of genetic research. These contemporary sequencing methods are much more sensitive, enabling clinicians and researchers alike to discover a greater volume of submicroscopic views of our genomes.

NGS platforms are able to sequence millions of small fragments of DNA in parallel. Researchers can choose a wide view to look at multiple samples, or zoom in for an in-depth analysis of a small, specific area of interest. Massive amounts of DNA sequencing data can be generated quicker, and more cost-effective than ever before.

The latest NGS technology can be applied for multiple applications for the best results; from short to long reads, from DNA to RNA, and from epigenome to metagenome. Sequencing enables researchers to uncover valuable genetic information that expands future discoveries.

Macrogen offers you peace of mind with 20+ years of experience in genomics. Simply enjoy high quality data & affordable prices!

Whole Genome Sequencing

Whole genome sequencing (WGS) is the process of determining the complete DNA sequence of an organism's genome, and the most comprehensive tool for detecting single nucleotide variants, insertions, deletions, copy number changes and structural variants. Macrogen’s WGS service is available for all species.

WGS can identify and isolate genetic variations, which is why it is commonly used for research purposes. Today, WGS is being widely applied in the fields of agriculture, infectious disease, immunology, cancer research and many more, and it’s yielding new insights surrounding evolutionary biology, various inherited conditions and drug development.

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq X Ten / HiSeq 4000 / HiSeq 2500
  • Illumina MiSeq
  • PacBio Sequel II / Sequel / RS II

Data Analysis

Whole Genome de novo Sequencing

De novo sequencing offers a way to uncover information about the entire genome of microorganisms or animals and plants that are not known. It assembles long parallel phases of “novel” genetic material, meaning it codes new DNA without referencing an existing amino acid sequence. This type of whole genome sequencing is highly accurate and can replicate or complete a blueprint for complex or polyploid genomes.

This primary generation of genetic material is used to spot chromosome deletions, inversions, or translocations, which may indicate the likelihood of a genetic disorder. Researchers use a variety of data analysis methods to obtain information from sequenced stretches of DNA.

Standard Data Analysis
  • Genome Survey (K-mer Analysis)
  • Consensus Sequence (Reads assembly into contigs)
Advanced Data Analysis
  • Gene Prediction
  • Gene Annotation

Whole Genome Resequencing

Resequencing a genome implies the comparison of a common genetic sequence to the newly sequenced data. This type of whole genome sequencing can scan or test for specific genetic variations. Any mutations of the DNA from the standard reference sample will enable new discoveries in a research setting and detection of a patient’s potential for disorders in the clinical setting. There are several methods of data analysis that follow resequencing, including:

Standard Data Analysis
  • Alignment (Read mapping to current reference genome)
  • Variant Calling (SNPs, InDels)
  • CNV, SV Analysis* (Copy number variation, Structural variation)
Advanced Data Analysis
  • A variety of options for mapping algorithms, variant detection algorithms, annotations, mapping to public database, group analysis, and case-control analysis are available.

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Whole Exome Sequencing

Whole exome sequencing (WES) is a targeted approach that makes possible to identify variations in the protein coding part in a genome. For human, only about 2% of the whole genome is made up with exomes. This can be especially valuable because most known genetic variants actually occur in exons. With whole exome sequencing, DNA of known organism is mapped out using a reference genome, and specific genetic mutations and disease-causing variants are identified.

WES method can provide the genetic basis of Mendelian diseases or traits that have proven intractable to conventional gene discovery strategies. Exome sequencing of parent–child trios is a highly effective approach for identifying de novo coding mutations of a disease, as multiple de novo events occurring within a specific gene (or within a gene family or pathway) is an extremely unlikely event.

Commercially available target enrichment kits mainly utilize hybridization capture method using magnetic beads. Macrogen uses several industry leading kits for WES service depending on researchers’ needs. Please get it touch with us if you are looking to conduct other species’ exome experiment.

Main Capture Kits (Human)

  • Agilent SureSelect Human All Exon kit
  • Illumina Truseq DNA Exome Kit
  • Twist Biosciences Human Core Exome Kit

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500

Data Analysis

Standard Data Analysis

  • Alignment (Reads mapping to current reference genome, hg19 or hg38)
  • Variant Calling (SNPs, InDels)
  • Annotation to various databases

Advanced Data Analysis

  • CNV (Copy Number Variation)
  • Cancer Analysis, Family/Trio Analysis, Population Analysis
  • A variety of options for variant calling pipeline are available.

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Targeted Panel Sequencing

Targeted sequencing can be extremely cost-effective, while providing more in depth view of the genes of interest. Computing time for anlayzing targeted sequencing data takes less time, as it usually targets focused number of genes that are known to be related to a disease.

Macrogen provides various enrichment methods, as well as helps customization of a panel for your genes of interest. DNA from Tissue, Formalin-Fixed paraffin-Embedded (FFPE), Blood, Cell Cultures, Clinical Samples and Fine Needle Aspirates(FNA) can be used.

Cancer Panel Sequencing characterizes fast-track oncology data by surveying  either hotspot mutations or whole exome sequences of oncogenes and tumor suppressor genes.  

Enrichment Methods

  • PCR based amplification
  • Hybridization capture

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500
  • Illumina MiSeq
  • ThermoFisher Ion Torront PGM / Proton

Data Analysis

Standard Data Analysis

  • Alignment (Read mapping to current reference genome)
  • Variant Calling (SNPs, InDels)
  • Annotation to various databases

Advanced Data Analysis

  • Population Analysis
  • A variety of options for variant calling pipeline are available.

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Transcriptome Sequencing

A transcriptome is a collection of all the transcripts present in a given time point. The sequence of an RNA mirrors the sequence of the DNA from which it was transcribed. Consequently, by analyzing the entire collection of RNAs or transcriptomes, researchers can determine when and where each gene is turned on or off in the cells and tissues of an organism.

Whole Transcriptome Sequencing (WTS) or RNA sequencing can give researchers more visibility into changes that can occur in certain time points or states of diseases. Researchers can also see more details about how a transcriptome reacts to certain drugs, environmental situations or other conditions.

mRNA / mRNA + lncRNA Sequencing

This type of RNA sequencing is one of most popular methods for analyzing disease states and biological processes in the study of transcriptomes. This method is extremely in-depth, allowing researchers to find new genes that were previously imperceptible.

Library Preparation Methods

Enrichment
  • Poly A enrichment (e.g. TruSeq Stranded mRNA)
  • rRNA depletion (e.g. TruSeq Stranded Total RNA with Ribo-Zero)
cDNA Synthesis
  • Random hexamer priming (e.g. TruSeq mRNA)
  • Poly A priming (e.g. SMARTer Ultra Low Input RNA)
Strand Orientation
  • Strand-specific approach provides gene expression information with relation to strand orientation.

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500

Data Analysis

Transcriptome de novo Sequencing

cDNA sequence reads are assembled into transcripts by a transcript assembly algorithm without the aid of a reference genome.

Standard Data Analysis
  • Assembly
  • Gene Expression Profile
Advanced Data Analysis
  • BlastNT or BlastNR
  • Differentially Expressed Genes (DEGs)
  • Gene Annotations & Ontology Analysis
Transcriptome Resequencing

cDNA sequence reads are aligned to a reference genome. Availability of this analysis pipeline is dependent on the completeness of a reference.

Standard Data Analysis
  • Alignment (Read mapping to current reference genome)
  • Gene Expression Profile
  • SNP calling (for Human, Mouse, Rat)
  • Novel Transcripts
  • Alternative Spliced Transcripts
  • Fusion Gene (for Human, Mouse, Rat)
Advanced Data Analysis
  • Differentially Expressed Genes (DEGs)
  • Gene Ontology Analysis
  • Customized Analysis

Small RNA Sequencing

This type of sequencing targets and isolates small RNAs (up to 200nt), including microRNA (21-23nt), by targeting the size range to discover how non-coding RNA affects different types of gene expressions. Small RNAs are known to be involved in regulation of gene expression in cellular process. Macrogen's NGS technology is able to sequence and quantify all the small RNA families in a sample and profile the miRNA, siRNA, piRNA and other non-coding RNAs.

Sequencing Platforms

  • Illumina HiSeq 4000 / HiSeq 2500

Data Analysis

Standard Data Analysis
  • Expression Profiles of small RNA (miRNA, tRNA, piRNA, snoRNA, snRNA etc.)
  • Novel miRNA
  • Categories of small RNA
Advanced Data Analysis
  • Target Prediction of Known and Novel miRNA
  • Gene Set Analysis
  • Differentially expressed miRNA

Extracelluar RNA / Exosomal RNA Sequencing

Extracellular RNA or exosomal RNA, which exists outside cells in living fluids such as plasma or urine, can be used in various studies such as liquid biopsy through analysis of various transcription bodies (miRNA, piRNA, snRNA, snRNA, snRNA, and mRNA).

Sequencing Platforms

  • Illumina HiSeq 4000 / HiSeq 2500

Data Analysis

Standard Data Analysis
  • RNA composition analysis
  • Expression profiles of smRNA and mRNA
  • Novel miRNA prediction
Advanced Data Analysis
  • Differentially expressed small RNAs
  • Customized analysis

Iso-seq

By creating a full-length transcript, you can characterize the transcript isoform across the target gene or transcriptome. Specifically, it provides accurate information about the alternately spliced exon and transcription start site (TSS).

Library Preparation Kit

  • PacBio Iso-Seq™ Express Template Preparation

Sequencing Platforms

  • PacBio Sequel II / Sequel / RS II

Data Analysis

  • Transcript isoform discovery and their validation
  • Coding and annotation with SNPs
  • Profiling and characterization junctions
  • Quantitative determination and identification of regulatory transcripts

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Single Cell Sequencing

If traditional transcriptome sequencing (also called bulk RNA sequencing) is the way to see the average gene expression in different cells, then Signal Cell RNA Sequencing can analyze thousands of transcription bodies simultaneously at a single cell level, so we can see the difference in gene expression values for different cell types. In addition, a full-length (clonotype level) analysis of the B cell, T cell is possible.

Library Preparation Kit

  • 10X Genomics Chromium 5'GEX
  • 10X Genomics Chromium 3'GEX
  • 10X Genomics Chromium V(D)J
  • 10X Genomics Chromium ATAC

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500

Data Analysis

Standard Data Analysis

  • Gene expression and cell clustering analysis
  • Simultaneous single cell analysis of gene expression
  • Immune profiling and cell surface protein expression

Advanced Data Analysis

  • Sample aggregation
  • Gene expression and cell clustering analysis using Seurat
  • Cell identification
  • Differentially Expressed Genes (DEGs) analysis
  • Gene ontology analysis
  • Trajectory analysis

Single Cell Immune Profiling

  • Paired full length V(D)J gene expression identification
  • Clonality and diversity of TCR and/or BCR

Single Cell Surface Protein Profiling

  • Surface protein expression and cell clustering analysis

Single Cell ATAC Sequencing

  • Chromatin accessibility profiles and cell clustering analysis

Single Cell CNV Profiling

  • Chromatin accessibility profiles and cell clustering analysis

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Epigenome Sequencing

Epigenome sequencing confirms that the function of a certain gene is expressed by external environmental factors. Methylation of DNA and acetylation of histone protein are the most representative epigenetic control mechanisms for the transcription of genes. They can be analyzed more effectively with NGS technology.

Comparison of Epigenomic Application

Application
Bisulfite Conversion
Advantages
WGBS
High resolution
Methyl-Cap
Methylated CpG enrichment

Whole Genome Bisulfite Sequencing

Whole Genome Bisulfite Sequencing (WGBS) is a method of observing the degree of DNA methylation by treating DNA using sodium bisulfate, which converts non-methylated cytosine to uracil, and analyzing the sequence.

Methyl-capture Sequencing

Methyl-Capture Sequencing is a method that captures the parts of the CpG where methylation waspreviously reported and analyzes the sequence converted to sodium sulfite to observe the methylation degree.

Sequencing Platforms

  • Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500

Data Analysis

Standard Data Analysis

  • Global methylation profiles
  • Specific methylation profiles e.g. CG islands / Differentially Methylated Regions (DMRs)

Advanced Data Analysis

  • DMR-associated genes
  • Gene set analysis
  • Comparative analysis

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

Metagenome Sequencing

Metagenome sequencing is a method of identifying microbial communities in different environments including the ones that cannot be cultured in laboratory setting. It is used primarily to analyze the distribution and types of bacteria and fungi, and to identify their interactions and roles.

It is applied to various areas such as monitoring microbial clusters through environmental samples such as soil and sea water, and analysis of fermented food, environmental wastewater and lactobacillus. Human gut microbiome became a hot topic for scientific community since recent studies revealed various relations to human diseases and conditions. The number of genes in intestinal microorganisms is about 150 times greater than human genes.

Sequencing Platforms

  • Metagenome Amplicon Sequencing : MiSeq / HiSeq2500
  • Metagenome Shotgun Sequencing : HiSeqX / NovaSeq
  • Full length 16S rRNA Sequencing : PacBio RSII / Sequel

Data Analysis

Amplicon Metagenomic Sequencing

Amplicon metagenomic sequencing enables efficient identification of microorganism diversity using conserved genes (16S, 18S, ITS, CO1, etc.) in a specific environment.

Standard Data Analysis
  • Community diversity analysis (Representative sequences, OTUs)
  • Probiotics analysis
Advanced Data Analysis
  • Phylogenetic tree
  • Hierarchical taxonomy graph
  • Heatmap
  • PCR biplot

Shotgun Metagenome Sequencing

Shotgun metagenomic sequencing is a comprehensive method of identifying all genes in all microorganisms present in a given environmental sample. The method enables to evaluate the microbial diversity and detect the abundance of species under different conditions. For shotgun sequencing, DNA is purified and randomly sheared into smaller fragments before sequencing. It provides information of which organisms are present and what metabolic processes are possible in the community.

Standard Data Analysis
  • Assembly
  • Gene prediction & annotation
  • Taxonomy analysis

*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.

NGS Sample Submission Guide

Overall Workflow

NGS Workflow.

Sample Preparation

Start by checking requested input amount from our QC criteria.

DNA

  • Quantify your sample using fluorescent based method.
  • Check quality of your sample by gel electrophoresis.
  • Aliquot your DNA sample in nuclease free water or TE buffer in a clearly labeled 1.5~2.0ml microcentrifuge tube sealed with parafilm tightly. Refer to our packing guideline.

RNA

  • Quantify your sample using fluorescent based method.
  • Check quality of your sample by BioAnalyzer or equivalent.
  • Optimal condition for RNA storage and/or delivery is ethanol precipitation method (stable for 1 year at -20C) Alternatively, RNA transport reagent from various commercial companies can be used. e.g. Ethanol Precipitation
  • Aliquot your RNA sample in nuclease free water in a clearly labeled 1.5~2.0ml microcentrifuge tube sealed with parafilm tightly. Refer to our packing guideline. If dry ice shipping is needed, please contact your Macrogen representative.

Include a brief description of extraction and/or enrichment protocol used in the “Comments” fields of the order sheet.

Certain carriers and pellet paint types are not compatible with aspects of our sequencing technology.

Non-robust results have been observed from samples where Lithium Chloride (LiCl), fluorescent pellet paint, or tRNA was used prior to precipitation and resuspension for sample preparation input.

Shipping Samples to Macrogen

Place sealed individual microcentrifuge tubes in a 50ml disposable screw cap tube for additional insulation during shipment. To prevent sample tubes from moving during shipment, pack any remaining space in the 50ml tube with clean tissue paper prior to sealing. Refer to our packing guideline.

Contact your Macrogen representative to get access to our online shipment system. Following should be included in the package.

  • Prepared sample tubes
  • Adequate cooling agent e.g. ice pack, dry ice
  • Cushioning material e.g. bubble wrap, foam, tissue paper
  • Printed Macrogen order sheet

Print the 3x shipping labels and 3x commercial invoices, sign and enclose with your shipment and contact your local office of chosen courier to schedule a pick-up and dispatch for the same day.

Your sample and data will be discarded after 3 months of data delivery. You may extend the period with a special request.