1. Next Generation Sequencing
Miluše Hroudová
Laboratory of Genomics and Bioinformatics
Institute of Molecular Genetics of the ASCR, v.v.i.
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

2. Basic Terms
Base-pair - basic building block of double-stranded DNA, unit of DNA segment length (bp)
Read - continuous sequence produced by sequencer
Coverage - the number of short reads that overlap each other within a specific genomic region (how many times the particular base or region is read)
Consensus sequence - idealized sequence in which each position represents the base most often found when many sequences are compared
Contig - set of overlapping segments (reads) of DNA sequences forming continuous consensus sequence
Assembly - aligning and merging fragments of DNA sequence (reads, contigs) in order to reconstruct the original sequence
Scaffold - set of linked non-contiguous series of genomic sequences, consisting of contigs separated by gaps of known length
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

3. Next Generation Sequencing (NGS) intro
Modern high-throughput DNA sequencing technologies
Massive, parallel, rapid ...
Decreasing price, time, workflow complexity, error rate
Increasing data quantity and quality, read lenght (data storage capacity), repertoire of bioinformatics tools
Wide range of applications
Third Generation Sequencing (single molecule, real time, in situ ...)
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

4. Input Material, Target Sequence
DNA
De novo genome seq
Resequencing (ChIP-Seq)
Amplicon seq (16S)
Sequence capture
Base modification detection
Genomic variations
eukaryotic
viral
prokaryotic
=> Genomics
chromosome l
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

5. Genomics
Area of genetics that concerns the sequencing and analysis of an organism’s genetic information
DNA sequencing + bioinformatics => sequence, assemble and analyze the function and structure of genomes (the complete set of DNA within a single cell of an organism)
Bacterial genome
Human genome

6. Input Material, Target Sequence
RNA
RNA Seq (Whole Transcriptome
Shotgun Seq – WTSS, normalized)
SNPs detection
RNA species other than mRNA
Quantitative seq
(without normalization)
Total RNA
Coding RNA
4 % of total
Functional RNA
96 % of total
Pre-mRNA
(hnRNA)
mRNA
Pre-rRNA
Pre-tRNA
snRNA
snoRNA
miRNA
siRNA
All organisms
Eukaryotes only
rRNA
tRNA
=> Transcriptomics
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

7. DNA sequencing procedure
Transcriptomics
Study of the transcriptome - the complete set of RNA transcripts produced from the genome, under specific circumstances at particular place and time
Methods: RT PCR, Microarrays, mRNA seq
mRNA sequencing procedure
Total RNA
mRNA
Fragmented mRNA
cDNA library
cDNA
Raw data (reads)
polyA
mRNA selection
rRNA depletion
Temperature based
fragmentation
Reverse
transcription
Library
preparation
Adapter ligation
Size selection
Sequencing run
Normalized cDNA
Normalization
Optional
DNA sequencing procedure
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

8. RNA Quality
quality of the starting total RNA - RNA integrity number (RIN)
RIN<7 => unequal read distribution along 5’ and 3’ ends
=> bad sequencing results
Number of reads
454 reads distribution
RIN < 7
RIN > 9
Agilent Bioanalyzer traces

9. cDNA synthesis Total RNA (ug) mRNA with polyA 3’end
SMARTer II A Oligo:
5’-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3’
Modified CDS Primer
5’-AAGCAGTGGTATCAACGCAGAGTTTTTGTTTTTTTCTTTTTTTTTTVN-3’
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

10. cDNA normalization TRIMMER cDNA normalization kit (Evrogen)
abundant transcripts
rare transcripts
TRIMMER cDNA normalization kit (Evrogen)
DSN = duplex-specific nuclease
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

11. Sequencing Principles
Sequencing by Synthesis
Sanger/Dideoxy chain termination (Life Technologies, Applied Biosystems)
Pyrosequencing (Roche/454)
Reversible terminator (Illumina )
Ion proton semiconductor (Life Technologies)
Zero Mode Waveguide (Pacific Biosciences)
Sequencing by Oligo Ligation Detection
SOLiD (Applied Biosystems)
Other
Asynchronous virtual terminator chemistry - HeliScope (Helios)
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

12. Actual Sequencing Platforms
Roche/454 (GS FLX+/GS Junior)
Illumina Genome Analyzer (HiSeq/MiSeq/NextSeq)
Life Technologies (3500 Genetic Analyzer,
Ion Torrent Proton/PGM)
Pacific Biosciences (PACBIO RSII)
Applied Biosystems (SOLiD, 3730xl DNA Analyzer )
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

13. Sanger (3500 GA, 3730xl DNA Analyzer)
Sequencing by synthesis
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

14. Oligo Ligation Detection (SOLiD)
Sequencing
by ligation
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

15. Reversible Terminator (HiSeq, MiSeq, NextSeq)
Cluster generation on a flow-cell surface
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

16. Reversible Terminator (HiSeq, MiSeq, NextSeq)
Sequencing by synthesis
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

17. Pyrosequencing (GS FLX, GS Junior)
Sequencing by synthesis
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

18. Pyrosequencing (GS FLX, GS Junior)
Sequencing by synthesis
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

19. Sequencing Matrices Sanger, 96-well, 8 capillaries 96 x 600 bp / 24 h
1400 €
Pyrosequencing, 2 regions
1,000,000 x 600 bp / 20 h
5500 €
Revers. terminator, MiSeq
10,000,000 x 250 bp / 40 h
1150 €
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

20. General Workflow Nucleic acid isolation/purification
RNA – selection of particular RNA species, cDNA synthesis
DNA – fragmentation, size selection (shotgun x paired end)
Seq library preparation (platform specific adaptors ligation, indexes)
Amplification of seq library (DNA-binding beads and other carriers)
Sequencing run set up
Image processing (images => sequence + quality information)
Data analysis (assembly, mapping, annotation ...)
Special tricks for amplicons, SeqCap, ChIP-Seq, small RNAs ...
user
service
service
user
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

21. Pyrosequencing workflow
Library preparation:
Adaptor ligation
Fragmentation
Emulsion PCR amplification:
Bead deposition onto PicoTiter Plate (PTP):
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

22. Paired-end x Mate-pair
Paired-end – sequencing from both fragment ends (< 1 kb)
Mate-pair – longer (3-20 kb) molecules circularized via internal adapter x

23. Mate-pair types
Mate-pair – longer (3-20 kb) molecules circularized via internal adapter
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

24. Parametres Comparison
PacBio RSII
Sequencing by
synthesis
> 4000 bp
99,999%
30 Min – 3 Hours
1.6 GB
Read length, fast,
no amplification,
real time record
0.06 M
Low throughput,
low accuracy
Liu et al Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

25. Parametres Comparison
Liu et al Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

26. Parametres Comparison of Benchtop Variants
Junior
700 bp
70 Mb
18 hours
2 days
Pyrosequencing
Minimize hand on time,
increase emPCR reproducibility
On/Off instrument µg
Liu et al Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

27. Applications and Suitable Seq Type
de novo DNA/RNA seq – Illumina, Roche/454 (PE), PacBio
Resequencing – SOLiD, Illumina
SNPs detection – Roche/454, PacBio (x InDels variation – Illumina, SOLiD)
Sequence capture - Illumina
Sanger - low-coverage sequencing of individual positions and regions (e.g., diagnostic genotyping) or the sequencing of virus- and phage-sized genomes
Ion Torrent – short amplicons
SOLiD - quantitative applications, small RNAs, epigenomics
HeliScope – quantitative applications
Combination of methods
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

28. Data Analysis, Assembly, Annotation
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

29. Data Analysis, Assembly, Annotation
technology compatible software (user friendly, inefective)
general, free access software (search for optimal tool)
user developed (lack of qualified bioinformaticians)
combination of different platforms data x problems with assemblers
platform specific errors, incompatible software parametres
multiple data filtering procedures
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

30. Machine/Service Availability
IMG – Roche/454 GS FLX+ (full run including library prep 5500 €/0,7GB)
- Illumina NextSeq (next year? )
Illumina MiSeq – IEM AS CR, GeneCore EMBL (1150 €/ 10 GB)
Illumina – GeneCore EMBL (HiSeq lane 100 bp PE 2500 €/200 GB)
Ion Torrent - GeneCore EMBL, TU Liberec
PacBio –Netherlands (Macrogen), Germany, Switzerland
Beijing Genomics Institute (BGI, China) – Illumina HiSeq 2000
- Roche GS FLX+
- SOLiD 4
- Ion Torrent
- Sanger 3730xl DNA Analyzer
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

31. Our Sequencing Projects
GS FLX+, Roche 454
HiSeq2000/MiSeq, Illumina
Amplicon seq
(environmental samples,
16S rDNA genes)
De novo genome sequencing
(bacteria, protozoa, platyhelminthes, plants ...)
Sequence capture
(human cancer research,
animal population genetics ...)
Metagenomics
(simple bacterial consortia
x complex environmental samples)
Transcriptomics
(protozoa, cnidarians, insects,
human cancer research ...)
Beckman CEQ 2000XL
- minor sequencing analyses
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

32. Transcriptomics (Evo-Devo Studies)
Craspedacusta sowerbyi
Six and Pou genes
early evolution
Hroudova et al PLoS ONE, 7(4): e36420

33. Sequencing Hot Today and Near Future
Single-Molecule Real-Time seq – SMRT Pac Bio
(without amplification necessary for signal detection)
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

34. Sequencing Hot Today and Near Future
Single cell DNA/RNA seq based on micro/nanofluidics technology
(without WGA based on MDA - Φ29 DNA polymerase)
Nanopores
(reduced enzymatic steps,
electric current based detection)
Silicon based nanopores (IBM)
Human genome (30x) under 1000 $ already announced by Illumina (HiSeq X Ten)
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

35. Before You Start Planning Seq Experiment
sufficient sample source
targeted application/platform
computational capacity (storage, back up, operations)
bioinformatics support
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

36. Take-away message
NGS - high-throughput, massive, parallel, rapid DNA sequencing
Third generation – single molecule, real time, reduced chemistry
Basic NGS principles – synthesis, ligation
Basic workflow
sample - fragmentation - library prep - seq run - data analysis
Applications – de novo seq, reseq, amplicons, SeqCap, RNA seq (quantitative versus normalized)
Choose the right one application and prepare sample appropriately
Basic data analysis pipeline
image acquisition, quality metrics - filtering - contig building - annotation
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”

37. of Genomics and Bioinformatics
Acknowledgement
Laboratory
of Genomics and Bioinformatics
IMG AS CR, Prague
Čestmír Vlček
Václav Pačes
Jan Pačes
Hynek Strnad
Michal Kolář
Jakub Rídl
Šárka Pinkasová
Laboratory of Transcriptional Regulation, IMG (Dr. Zbyněk Kozmik)
Core facility of Genomics and Bioinformatics, IMG (Mgr. Šárka Kocourková, Mgr. Marcela Vedralová)
GeneCore, EMBL, Heidelberg (Dr. Vladimír Beneš)
Roche CR (Diagnostic Division), Genetica CR (Illumina Division)
The presentation is supported from the project OP EC CZ.1.07/2.3.00/ “Founding the Centre of Transgenic Technologies”