Introduction
Features of First- and Next-Generation Sequencing
Platform | Throughput | Length | Quality | Cost | Applications | Sources of error | Advantages | Disadvantages |
---|---|---|---|---|---|---|---|---|
Sanger
| 6 Mb/day | 1,000 nt | 10-4-10-5 | ~$500/Mb | Small sample sizes, genomes, SNPs, long haplotypes, low complexity regions, etc. | Polymerase/amplification, low intensities/missing termination variants, contaminant sequences | Longest reads, gold standard for validations | High cost, low throughput |
454/Roche
| 750 Mb/day | 400 nt | 10-3-10-4 | ~$20/Mb | Complex genomes, SNPs, structural variation, indexed samples, small RNAs, mRNAs, etc. | Amplification, mixed beads, intensity thresholding, homopolymers, phasing, neighbor interference | Longer reads, easier to assemble | Medium throughput, expensive, indel errors more likely |
Illumina
| 5,000 Mb/day | 100 nt | 10-2-10-3 | ~$0.50/Mb | Complex genomes, counting (SAGE, CNV Chip, small RNA), mRNAs, structural variation, bisulfite data, indexing SNPs, etc. | Amplification, mixed clusters/neighbor interference, phasing, base labeling | Lower cost, widely adopted platform, most well-developed bioinformatics efforts | Higher base substitution error rate, shorter reads |
SOLiD | 5,000 Mb/day | 75 nt | 10-2-10-3 | ~$0.50/Mb | Complex small genomes, counting (SAGE, ChiP, small RNA, CNV), SNPs, mRNAs, structural variation, indexing, etc. | Amplification, mixed beads, phasing, signal decline, neighbor interference | Lower cost, 2-base encoding chemistry, higher per-base accuracy | Shortest read lengths, still an emerging platform |
First generation | Second generation | Third generation | |
---|---|---|---|
Fundamental technology
| Size-separation of specifically end-labeled DNA fragments | Wash-and-scan SBS | Single molecule real time sequencing |
Resolution
| Averaged across many copies of the DNA molecule | Averaged across many copies of the DNA molecule | Single DNA molecule |
Current raw read accuracy
| High | High | Lower |
Current read length
| Moderate (800-1000 bp) | Short (generally much shorter than Sanger sequencing) | > 1000 bp |
Current throughput
| Low | High | High |
Current cost
| High cost per base, Low cost per run | Low cost per base, High cost per run | Low cost per base, High cost per run |
RNA-sequencing method
| cDNA sequencing | cDNA sequencing | Direct RNA sequencing |
Time to result
| Hours | Days | < 1 day |
Sample preparation
| Moderately complex, PCR amplification is not required | Complex, PCR amplification is required | Various |
Data analysis
| Routine | Complex (due to large data volumes & short reads) | Complex |
Primary results
| Base calls with quality values | Base calls with quality values | Base calls with quality values |
Genomics in Human Disease: Whole, Exome, and Transcriptome Sequencing
Distribution of Clinical Trials using Genomic Sequencing
Types of Clinical Studies
Condition | Diseases |
---|---|
Bacterial and Fungal Diseases
| Mycoses, Osteitis, Pelvic Infection, Pelvic Inflammatory Disease, Proteus Infections |
Blood and Lymph Conditions
| Anemia, Blood Coagulation, Burkitt Lymphoma, Hodgkin Disease, Hemorrhagic, Hemoglobinopathies, Leukemias, Lymphomas, Lymphoproliferative Disorders, Multiple Myeloma |
Cancers and Other Neoplasms
| Adenocarcinoma, Neuroblastoma, Nevus, Osteosarcoma, Retinoblastoma, multiple neoplasms, carcinomas, etc. |
Digestive System Diseases
| Digestive System Neoplasms, Duodenal Diseases, Gastroenteritis, Ileal, Jejunal, Stomach Diseases and Neoplasms |
Diseases and Abnormalities at or before Birth
| Multiple congenital anomalies, Cardiovascular Anomalies/Congenital Heart Disease, Inborn Diseases, Hemoglobinopathies, Neurocutaneous Syndromes, Neurofibromatoses |
Ear, Nose, and Throat Diseases
| Deafness, Hearing Disorders, Hearing Loss |
Eye Diseases
| Retinoblastoma |
Gland and Hormone Related Diseases
| Acromegaly, Endocrine Disorders, Dwarfism, Neoplasms, Hyperparathyroidism, Parathyroid and Pituitary Diseases |
Heart and Blood Diseases
| Aortic Valve Stenosis, Arterial Occlusive Diseases, Cardiomyopathies, Coronary Artery Disease, MI |
Immune System Diseases
| AIDS, Lymphomas, Hodgkin Disease, Immunoproliferative Disorders, Leukemias, Myelomas, Mycosis, Macroglobulinemia |
Muscle, Bone, and Cartilage Diseases
| Acromegaly, Bone Diseases, Dwarfism, Congenital Limb Anomalies, Musculoskeletal Abnormalities, Osteitis |
Nervous System Diseases
| ALS, Aphasias, Brain Neoplasms, CNS Diseases, Coma, Communication Disorders, Deafness, Dementia/Delirium, Motor Neuron Diseases, Neurocutaneous Syndromes, Neurodegenerative Diseases, Neurofibromas/NF, Neuromuscular Diseases, Pain, Speech Disorders, Spinal Cord Diseases |
Skin and Connective Tissue Diseases
| Breast Diseases/Neoplasms, Neurocutaneous Syndromes |
Symptoms and General Pathology
| Coma, Communication, Deafness/Delirium, Hearing Disorders, Hemolysis, Inflammation, Ischemia, Neurobehavioral, Pain, Sclerosis, Sepsis/Shock |
Urinary Tract, Sexual Organs, & Pregnancy
| Adnexal Diseases, Renal Cell Carcinoma, Endometritis, Kidney Diseases, Pelvic Inflammatory Disease, Prostatic Neoplasms, Urogenital Neoplasms, Uterine and Urologic Diseases, Wilm's Tumor |
Viral Diseases
| AIDS, Burkitt Lymphoma, HIV Infections |
Study Title/Sponsor | NCT #/ # Enrolled/ Start Date | Condition | Description |
---|---|---|---|
Next Generation to Identify Genetic Causes of Disease in Patients Participating in NICHD Clinical Protocols
NICHD
| NCT01375543 100 June 2011 | Genetic diseases (pediatric) | Use of DNA samples to conduct exome and genome sequencing |
Feasibility Clinical Study of Targeted and Genome-Wide Sequencing
University Health Network, Toronto
| NCT01345513 100 March 2011 | Solid Tumors | Targeted and genome-wide sequencing of DNA to enable molecular characterization of tumors. |
Biomarkers in Tissue Samples from Patients with High-Risk Wilms Tumor
NCI
| NCT01118078 100 March 2010 | Kidney Cancer | Application of array-based methods and NGS to identify candidate molecular targets |
Whole Genome Medical Sequencing for Genome Discovery
NHGRI
| NCT01087320 100 Feb 2010 | Congenital Syndromes/ Genetic Disorders | Using genomic sequencing to identify genetic causes of disorders that are difficult to identify with existing techniques |
Studying DNA in Tumor Tissue Samples from Patients with Localized or Metastatic Osteosarcoma
NCI
| NCT01062438 99 Jan 2010 | Sarcoma | Genomic expression profile in osteosarcoma tumor samples using transcriptome sequencing |
Genetics of Congenital Heart Disease
Nationwide Children's Hospital
| NCT01192048 1000 Dec 2009 | Congenital Heart Disease | Direct sequencing and/or microarray, whole-genome array comparative genomic hybridization (CGH) |
Integrated Whole-Genome Analysis of Hematologic Disorders
Stanford University
| NCT01108159 100 Sept 2009 | Hematologic Diseases | Whole-genome analysis/high-throughput sequencing using blood, bone marrow and skin biopsy samples |
Study of Tissue Samples from Patients with Lymphoma
NCI
| NCT00952809 300 March 2009 | Lymphoma; Small Intestinal Cancer | Generation of genome-wide maps of the distribution of nucleosomes and histone modifications as assessed by high throughput sequencing (ChIP-Seq) |
Genetics of Endocrine Tumours
Barts & The London NHS Trust
| NCT00461188 150 March 2007 | Acromegaly | Tumor samples studied using candidate gene sequencing |
DNA Analysis of Tumor Tissue Samples from Patients with Diffuse Brain Stem Glioma
St. Jude Children's Research Hospital
| NCT00899834 30 June 2006 | Brain & CNS Tumors | Genome-wide expression of RNA in tumor samples using gene expression profiling. Direct sequencing analysis of tumor DNA |
ClinSeq: A Large-Scale Medical Sequencing Clinical Research Pilot Study
NHGRI
| NCT00410241 2000 Dec 2006 | Cardiovascular Disease | Sequencing ~ 400 genes related to heart disease |
Laboratory Study of Lymphoblasts in Young Patients with High-Risk ALL
NCI
| NCT00896766 150 July 2006 | Leukemia | Pilot application of array-based methods and gene re-sequencing to identify candidate molecular targets for ALL |
Genome Expression in Lymphoma, Leukemia and Multiple Myeloma
NCI
| NCT00339963 3000 Nov 2001 | Lymphoma, Leukemia Multiple Myeloma | Participating centers send samples to the NCI for gene expression profiling, array-based comparative genomic hybridization and cancer gene re-sequencing. |