[A] knowledge of sequences could

contribute much to our understanding of

living matter.”
-Frederick Sanger
 First Generation: Sanger Sequencing

When to use Sanger Sequencing

• Sequencing single genes
• Sequencing 1-100 amplicon targets
• NGS confirmation
• Genotyping

BioEssays Volume 32, Issue 6, pages 524-536, 18 MAY 2010 DOI: 10.1002/bies.200900181
 What is NGS ?

 Next generation DNA sequencing (NGS) also known as deep-

sequencing
 Non-Sanger-based high throughput DNA sequencing
technologies.
 Compared to Sanger sequencing, NGS platforms
➢ Sequence as many as billions of DNA strands in parallel,

➢ Yielding substantially more throughput and

➢ Minimizing the need for the fragment-cloning methods that

are often used in Sanger sequencing of genomes.

Ozsolak, Fatih, and Patrice M. Milos.” Nature Reviews Genetics 12, no. 2 (February 2011) 87–98
 What can NGS do for you?

 Rapidly sequence whole genomes

 Zoom in to deeply sequence target regions (Hybrid selection,
amplicon sequencing)
 Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants
and splice sites, or precisely quantify mRNAs for gene expression
analysis
 Analyze epigenetic factors such as
➢ Genome-wide DNA methylation (Bisulphite seq) and

➢ DNA-protein interactions (Chip-Seq)

 Sequence cancer samples to study rare somatic variants, tumor sub

clones, etc
 Study microbial diversity in humans
 NGS Sequencing technologies

• Short read sequencing

• Long read sequencing
 Pyrosequencing

Youtube has
excellent
videos. Search
term:
Pyrosequencing

BioEssays Volume 32, Issue 6, pages 524-536, 18 MAY 2010 DOI: 10.1002/bies.200900181
 Illumina Reversible terminator chemistry

Excellent video
on youtube:
Illumina
Sequencing by
Synthesis

BioEssays Volume 32, Issue 6, pages 524-536, 18 MAY 2010 DOI: 10.1002/bies.200900181
 Applied Biosystem's SOLiD Sequencing by ligation

BioEssays Volume 32, Issue 6, pages 524-536, 18 MAY 2010 DOI: 10.1002/bies.200900181
 Asynchronous virtual terminator chemistry
performed by the HeliScope.

BioEssays Volume 32, Issue 6, pages 524-536, 18 MAY 2010 DOI: 10.1002/bies.200900181
 Oxford Nanopore

http://www2.technologyreview.com/news/427677/nanopore-sequencing/
Basic workflow for NGS library preparation

Head et al., BioTechniques 56, no. 2 (February 1, 2014)

 Things to consider before starting sequencing experiment

 What is the question you want to answer?

 Hypothesis- What sequence do you need for a specific aim?
 Hypothesis free- Sequence the whole genome
 How much data to generate?
 Sequencing result is the final answer or preliminary data?
 What are the costs of false -/+ relative to the cost of seq?
Example 1: Tumor/normal sequencing
 Requires very low false +/-
 High coverage, high variant calling stringency
Example 2: Microbial evolution
 Sequencing a drug resistant microbe to find functional changes
 High coverage, low variant calling stringency
Which factors influence the amount of data you generate?

 Number of samples (Biological/technical replicates, controls)

 Read type (Paired end Vs single read)
 Type of library
 Number of reads (Coverage)
 Read length
 Complexity of library
 Which sequencing machine to use
 Pooling with barcoding
 PCR-based artifacts
 RNA-Seq Sample Prep

Hunt AG. (2014) A Rapid, Simple, and Inexpensive Method for the Preparation of Strand-Specific RNA-Seq Libraries. Methods Mol Biol 1255:195-207
 RNA-Seq

 Strand-specific RNA-seq- Antisense transcription events

 Ligation of adaptors in a predetermined orientation
 Characterization of alternative splicing patterns (15-20%
of disease causing mutations affect splicing)
 Gene-fusion detection
 miRNA, snRNA, sRNA- Different sample preparation
methods
 Gene expression levels (disease vs normal; treatmetn vs
no treatment)
 Gene fusion

Ozsolak, Fatih, and Patrice M. Milos..” Nature Reviews Genetics 12, no. 2 (February 2011): 87–98.
DATA ANALYSIS
 General read processing

 Checking the overall success of the sequencing

process (number of filtered reads, number of
clusters)
 Remove rRNA artifacts (RNA Seq)
 Remove off-target sequences (Targeted sequencing)
 Trimming adapters
 Removal of PCR duplicates
 Convert the data into common file formats such as
FASTA, FASTQ

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 Mapping to a reference sequence Vs Denovo assembly

 Mapping to a reference sequence

 Different sequencing machines generate different
length
 Different aligner software available
 When strains or isolates are similar enough to a
reference sequence, reads can be mapped against
this reference by finding the unambiguous place
where an alignment generates the highest score for a
given read, similar to a BLAST search

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 De nov0 Assembly

 The aim of sequence assembly is to join short

sequences of nucleotides (sequence reads 35–1,000
bp in length) into contiguous sequences (contigs)
that represent the sequenced DNA.
 Sequence assembly is needed when no reference
genome is available, or when the sequenced DNA is
too different from a potential reference genome

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 What information can you get?

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 Variant detection

 Detecting SNVs –
 Search the mapped reads for bases that are different from the
reference sequence
 Sufficient coverage is required for confidence
 SNVs can be heterozygous
 Structural variants
 Can be detected by paired-end sequencing
 Estimate the distribution of fragment length
 Reads mapped to different chromosomes or have abnormal
distance, ordering or strand orientation

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 Insertion/Deletion

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 RNA Seq

 Different analysis procedures for longer RNA molecules (mRNA)

Vs small RNA (miRNA, snRNA)
 Classify the mapped reads into introns, exons, rRNA, intergenic
 Determine the expression levels of the annotated genes by
counting the reads mapped to the corresponding exon and then
divide by the length of the exon and total number of reads
(RPKM)
 To quantify different splicing isoforms
 Novel transcripts or isoforms

Gogol-Döring, Andreas, and Wei Chen, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-400-1_16.
 ChIP-Seq

 Use a “peak calling” tool to search for enriched regions in the ChIP-seq
data ChIP-seq data should be evaluated relative to a control data set
obtained either by sequencing the input DNA without ChIP or by using an
antibody with unspecific binding such as IgG

http://www.strand-ngs.com/features/chip-seq
 Single cell sequencing

 To study biological heterogeneity

 Cancer biology and oncology- Tumor heterogeneity
 Immunology0- TCR
 Microbiology- Metagenomics

Wang, Jian, and Yuanlin Song.” Clinical and Translational Medicine 6, no. 1 (December 2016).
 Single cell isolation methods

Wang, Jian, and Yuanlin Song.” Clinical and Translational Medicine 6, no. 1 (December 2016).
Alternative Genomic Strategies

Goodwin et al.,” Nature Reviews Genetics 17, no. 6 (May 17, 2016): 333–51
 References

 Buermans, H.P.J., and J.T. den Dunnen. “Next Generation Sequencing Technology: Advances and Applications.” Biochimica et Biophysica Acta
(BBA) - Molecular Basis of Disease 1842, no. 10 (October 2014): 1932–41. doi:10.1016/j.bbadis.2014.06.015.
 Gawad, Charles, Winston Koh, and Stephen R. Quake. “Single-Cell Genome Sequencing: Current State of the Science.” Nature Reviews Genetics 17,
no. 3 (January 25, 2016): 175–88. doi:10.1038/nrg.2015.16.
 Gogol-Döring, Andreas, and Wei Chen. “An Overview of the Analysis of Next Generation Sequencing Data.” In Next Generation Microarray
Bioinformatics, edited by Junbai Wang, Aik Choon Tan, and Tianhai Tian, 802:249–57. Totowa, NJ: Humana Press, 2012. doi:10.1007/978-1-61779-
400-1_16.
 Goodwin, Sara, John D. McPherson, and W. Richard McCombie. “Coming of Age: Ten Years of next-Generation Sequencing Technologies.” Nature
Reviews Genetics 17, no. 6 (May 17, 2016): 333–51. doi:10.1038/nrg.2016.49.
 Head, Steven R., H. Kiyomi Komori, Sarah A. LaMere, Thomas Whisenant, Filip Van Nieuwerburgh, Daniel R. Salomon, and Phillip Ordoukhanian.
“Library Construction for next-Generation Sequencing: Overviews and Challenges.” BioTechniques 56, no. 2 (February 1, 2014).
doi:10.2144/000114133.
 ———. “Library Construction for next-Generation Sequencing: Overviews and Challenges.” BioTechniques 56, no. 2 (February 1, 2014).
doi:10.2144/000114133.
 Kenneth Nelson, F., Michael Snyder, Andrew F. Gardner, Cynthia L. Hendrickson, Jay A. Shendure, Gregory J. Porreca, George M. Church, et al.
“Introduction and Historical Overview of DNA Sequencing.” In Current Protocols in Molecular Biology, edited by Frederick M. Ausubel, Roger Brent,
Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, and Kevin Struhl. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011.
doi:10.1002/0471142727.mb0700s96.
 Kircher, Martin, and Janet Kelso. “High-Throughput DNA Sequencing - Concepts and Limitations.” BioEssays 32, no. 6 (May 18, 2010): 524–36.
doi:10.1002/bies.200900181.
 Otto, Thomas D. “From Sequence Mapping to Genome Assemblies.” In Parasite Genomics Protocols, edited by Christopher Peacock, 1201:19–50. New
York, NY: Springer New York, 2015. doi:10.1007/978-1-4939-1438-8_2.
 Ozsolak, Fatih, and Patrice M. Milos. “RNA Sequencing: Advances, Challenges and Opportunities.” Nature Reviews Genetics 12, no. 2 (February 2011):
87–98. doi:10.1038/nrg2934.
 Papalexi, Efthymia, and Rahul Satija. “Single-Cell RNA Sequencing to Explore Immune Cell Heterogeneity.” Nature Reviews Immunology, August 7,
2017. doi:10.1038/nri.2017.76.
 Reuter, Jason A., Damek V. Spacek, and Michael P. Snyder. “High-Throughput Sequencing Technologies.” Molecular Cell 58, no. 4 (May 2015): 586–
97. doi:10.1016/j.molcel.2015.05.004.
 Wang, Jian, and Yuanlin Song. “Single Cell Sequencing: A Distinct New Field.” Clinical and Translational Medicine 6, no. 1 (December 2017).
doi:10.1186/s40169-017-0139-4.
 Useful links

 Single cell sequencing: https://www.illumina.com/content/dam/illumina-

marketing/documents/products/research_reviews/single-cell-sequencing-
research-review.pdf
 Annotations/Pathway analysis:
https://david.ncifcrf.gov
 Sequencing experiment design:
http://evomicsorg.wpengine.netdna-cdn.com/wp-
content/uploads/2015/06/Genomics-Study-Design-4_1_0.pdf
 Data visualization/analysis:
http://software.broadinstitute.org/software/igv/