The advent of NGS. Milestones in DNA sequencing. Very brief history of DNA sequencing

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1 Principles of Next Generation Sequencing (NGS) and applications to parasites Simone M Cacciò European Union Reference Laboratory for Parasites ISS, Rome EURLP Workshop, Rome May, 2016 Very brief history of DNA sequencing The first method involved a location-specific primer extension strategy established by Ray Wu at Cornell University in DNA polymerase catalysis and specific nucleotide labeling, both of which figure prominently in current sequencing schemes, were used to sequence the cohesive ends of lambda phage DNA. Frederick Sanger then adopted this primer-extension strategy to develop more rapid DNA sequencing methods at the MRC Centre, Cambridge, UK. He published a method for DNA sequencing with chain-terminating inhibitors in Walter Gilbert and Allan Maxam at Harvard also developed sequencing methods, including one for DNA sequencing by chemical degradation. Milestones in DNA sequencing 1985: The first free-living organism to have its genome fully sequenced was the Gram-negative bacterium Haemophilus influenzae in 1995 by The Institute for Genomic Research (TIGR). The circular chromosome contains 1,830,137 bases and its publication in Science marked the first published use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts. 1986: a worldwide effort produced the first complete eukaryotic genome, the yeast Saccharomyces cerevisiae 2000: the publication of the human genome was the principal event in the rise of genomics and consequently marks the beginning of the sequencing era. The initial human genome project had a cost of around 3 billion dollars. The advent of NGS In 2005, 454 Life Sciences introduced the Pyrosequencing technology, the first high-throughput technology that allowed the generation of thousands to millions of short sequencing reads in a single machine run. Since then, several other high-throughput platforms have been developed, such as Illumina, Solid, IonTorrent, and Pacific Biosciences. 1

2 Pyrosequencing: The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-out. Illumina: In this method, DNA molecules and primers are first attached on a slide or flow cell and amplified with polymerase so that local clonal DNA colonies, "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases (RT-bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides. Then the dye, along with the terminal 3' blocker, is chemically removed from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera. SOLID: Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. ION TORRENT: This method of sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template, the nucleotide is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. 2

Single molecule real time (SMRT) sequencing (Pacific Biosciences, PacBio): In this method, the DNA is synthesized in zero-mode wave-guides (ZMWs) small well-like containers with the capturing tools

The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.

3 Single molecule real time (SMRT) sequencing (Pacific Biosciences, PacBio): In this method, the DNA is synthesized in zero-mode wave-guides (ZMWs) small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand. This methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases. Different platforms, different chemistry Comparison of the different platforms Method Read length Accuracy (single read) Reads per run Time per run Cost / Mb Advantages Disadvantages Pyrosequencing (454) 700 bp 99.9% 1 million 24 hours $10 Long read size. Fast. Runs are expensive. Homopolymer errors. Sequencing by synthesis (Illumina) MiniSeq, NextSeq: bp; MiSeq: bp; HiSeq 2500: bp; HiSeq 3/4000: bp; HiSeq X: 300 bp 99.9% MiniSeq/MiSeq: 1-25 Million; NextSeq: Million, HiSeq 2500: 300 million - 2 billion, HiSeq 3/ billion, HiSeq X: 3 billion 1 to 11 days, depending upon sequencer and specified read length $ Potential for high sequence yield, depending upon sequencer model and desired application. Equipment can be very expensive. Requires high concentrations of DNA. Sequencing by ligation (SOLiD) bp bp 99.9% 1.2 to 1.4 billion 1 to 2 weeks $0.13 Low cost per base. Slower than other methods. Has issues sequencing palindromic sequences Ion semiconductor (Ion Torrent) up to 400 bp 98% up to 80 million 2 hours $1 Less expensive equipment. Fast. Homopolymer errors. SMRT (Pac Bio) 10,000-15,000 bp 87% 50,000 per SMRT cell, or megabases 30 minutes to 4 hours $0.13 $0.60 Longest read length. Fast. Detects 4mC, 5mC, 6mA Moderate throughput. Equipment can be very expensive. 3

4 Overview of the workflow of a NGS experiment Step 1: Planning the experiment Step 2: library preparation Step 3: sequencing Wet lab Step 1. Question and options Each question ideally requires a certain approach (library and seqencing platform,, and is more or less influenced by specific parameters. Some rules of thumbs are illustrated here below Step 4: data analysis Dry lab Step 2: wet lab Target organism(s) RNAseq WGS Total RNA extraction DNA extraction Sample preparation Sample preparation Next Generation Sequencing Data analysis Step 2: library preparation The DNA to be sequenced is used to construct a library of fragments that have synthetic DNAs (adapters) added covalently to each fragment end by use of DNA ligase. These adapters are universal sequences, specific to each platform, that can be used to polymerase-amplify the library fragments during specific steps of the process. The library fragments are amplified in situ on a solid surface, either a bead or a flat glass microfluidic channel, that is covalently derivatized with adapter sequences that are complementary to those on the library fragments. 4

5 Data analysis Raw sequence data from NGS Data analysis: a real challenge Trimming De novo assembly Contigs generation Protein databases queries Annotation Reference Mapping Conceptual translation Comparative genomics Of course, the collaboration between biologists and bioinformaticians is the solution. There are many web resources that provide data analysis (for free) Commercial software are also available Biological pathway search Virulence factors search NGS applications NGS in parasitology Next-generation sequencing can be used for Genome sequencing Genome resequencing Meta-genomics Transcriptome profiling (RNA-Seq) DNA-protein interactions (ChIP-sequencing) Epigenome characterization 5

Examples from our current research I will describe two research activities that are in progress in our laboratory and that will allow to discuss two NGS applications: 1) Comparative

Putignani Collaborators: Anna Rosa Sannella, Fabio Tosini, Elisabetta Manuali, Chiara Magistrali, Rune Stensvold, Semiramis Guimarães, Erica Boarato, Miles Beaman The COMPARE project

6 Examples from our current research I will describe two research activities that are in progress in our laboratory and that will allow to discuss two NGS applications: 1) Comparative genomics (COMPARE project) Collaborators: Anna Rosa Sannella, Giuseppe La Rosa 2) Metagenomics (Blastocystis and Dientamoeba) Collaborators: Nicola Segata, Francesco Beghini, Lorenza Putignani Collaborators: Anna Rosa Sannella, Fabio Tosini, Elisabetta Manuali, Chiara Magistrali, Rune Stensvold, Semiramis Guimarães, Erica Boarato, Miles Beaman The COMPARE project COMPARE is a large EU project with the intention to speed up the detection of and response to disease outbreaks among humans and animals worldwide through the use of new genome technology. Integrate state-of-the-art strategies, tools, technologies and methods for collecting, processing and analysing sequence-based pathogen data in combination with associated (clinical, epidemiological and other) data, for the generation of actionable information to relevant authorities and other users in the human health, animal health and food safety domains. 6

Our role within COMPARE We focus on protozoa (Cryptosporidium and Giardia) and the main objectives are: 1) To optimize the steps necesary for (oo)cyst purification from different matrices (mostly

bio-informatic pipeline(s) Purification of (oo)cysts This is necessary because (oo)cysts are always outnumbered by other organisms present in the sample.

7 Our role within COMPARE We focus on protozoa (Cryptosporidium and Giardia) and the main objectives are: 1) To optimize the steps necesary for (oo)cyst purification from different matrices (mostly stool) 2) To collect humas and animal samples of different geoographic origin, from both sporadic cases and outbreaks 3) To generate and analyze genomic sequences, and to establish ad hoc bio-informatic pipeline(s) Purification of (oo)cysts This is necessary because (oo)cysts are always outnumbered by other organisms present in the sample. Immunomagnetic separation of (oo)cysts from stools Enumeration of the (oo)cysts after double staining with monoclonal antibodies and DAPI How many (oo)cysts do we need? A single Cryptosporidium oocysts or a single Giardia cyst 15 contain about 40 femtograms (10- g) of genomic DNA The minimum amount of DNA for a NGS experiment is 1-2 nanograms, which in turn corresponds to 25-50,000 (oo)cysts. Since we cannot grow (easily) these parasites in the lab, what we have is what is in the sample. Also consider that any purification step will cause a reduction of the original number of organism. It follows that a sufficient amount of DNA could not be obtained from all samples. Genomic DNA versus Whole Genome Amplification A way to overcome this limitation is to submit the genomic DNA to Whole Gemome Amplification 23 kb 7

Is the DNA «clean» enough? It is important to check for enrichment of the target organism, but also to evaluate the presence of possible contaminants.

8 Is the DNA «clean» enough? It is important to check for enrichment of the target organism, but also to evaluate the presence of possible contaminants. We run a 16S-rRNA based PCR to detect the presence of bacterial contamination in the purified (or WGA-amplified) genomic DNA. Quality checks: -cloning and random sequencing -qpcr to detect increase in target DNA Samples examined Isolate Host Sample type Year Origin Cesium chloride IMS Ipochlorite VE1 Calf intestinal tissue 2015 Northern Italy yes yes ISSC6 Calf frozen oocyst 1996 Denmark yes yes TO1 Calf frozen oocyst 1990 Northern Italy yes yes C366 Goat kid feces in dicrhomate 2014 Southern Italy yes yes C320 Goat kid frozen feces 2013 Northern Italy no yes no C358 Calf frozen feces 2013 Central Italy no yes no C385 Goat kid fresh, unpreserved feces 2015 Southern Italy no yes no C386 Goat kid fresh, unpreserved feces 2015 Southern Italy no yes no C388 Lamb fresh, unpreserved feces 2015 Southern Italy yes no yes C389 Lamb fresh, unpreserved feces 2015 Southern Italy yes no yes Slo 1 Human fresh, unpreserved feces 2015 Slovenia no yes no Slo 2 Human fresh, unpreserved feces 2015 Slovenia no yes yes Results-1 Results-2 Isolate Total contigs N50 N75 Contigs to IOWA Sum of contigs to IOWA Isolate/IOWA % Isolate Bacterial 16S PCR Illumina Total reads N. reads to IOWA % reads to IOWA GC content VE1 negative 2x ,39 30,2 ISSC6 negative 2x ,79 30,3 TO1 weakly positive 2x ,86 36,9 C366 negative 2x ,12 31,2 C320 weakly positive 2x ,55 32,2 C358 positive 2x ,65 38,7 C385 negative 2x ,29 30,2 C386 positive 2x ,45 30,7 C388 negative 2x ,39 33,4 C389 negative 2x ,08 32,9 Slo 1 positive 2x ,13 39,0 Slo 2 negative 2x ,38 30,2 VE ,84 ISSC ,99 TO ,26 C ,14 C ,03 C ,23 C ,05 C ,01 C ,23 C ,84 Slo ,99 Slo ,11 8

selection search for highly polymorphic regions phylogenomics Metagenomics Metagenomics is defined as the direct genetic

9 All SNPs found in different isolates compared to chromosoem 1 in IOWA All non-synonymous SNPs in chr. 1 In progress Genome comparison, in particular: analysis of SNPs analysis of structural rearrangements search for genes under selection search for highly polymorphic regions phylogenomics Metagenomics Metagenomics is defined as the direct genetic analysis of genomes contained with a complex sample (environmental). There are two main experimental approaches: Amplicon sequencing Shotgun sequencing 9

Much less is known on the role played by eukaryotic organisms (protists, helminths) in the homeostasis of the human gut microbiome Why

10 Amplicon sequencing Shotgun sequencing Our question: protists in the human gut Blastocystis Many studies have focused on the human gut microbiome, with a particular emphasis on the bacterial component. Much less is known on the role played by eukaryotic organisms (protists, helminths) in the homeostasis of the human gut microbiome Why Blastocystis? 1) availability of reference genome sequences 2) high prevalence in humans, worldwide 3) controversial pathogenic role 4) many genetically different variants (subtypes) associated with human infection (9 STs) 10

Outline of the study A total of 2015 human gut microbiome samples from 12 metagenomic datasets were selected.

Results A pipeline was developed to detect Blastocystis sequences, and a detection threshold of >.

was detected in 262 samples (13% overall prevalence), of which 228 included a single subtype (62 ST2, 80 ST3, 29 ST4, 2 ST6, 3 ST7, 14 ST8 and 39 ST9), whilst 34

11 Outline of the study A total of 2015 human gut microbiome samples from 12 metagenomic datasets were selected. The original studies included subjects from different geographic origins and/or different disease conditions. Results A pipeline was developed to detect Blastocystis sequences, and a detection threshold of >.10 (meaning that at least 10% of the genome is represented) was chosen. Overall, Blastocystis spp. was detected in 262 samples (13% overall prevalence), of which 228 included a single subtype (62 ST2, 80 ST3, 29 ST4, 2 ST6, 3 ST7, 14 ST8 and 39 ST9), whilst 34 showed evidence for the presence of multiple subtypes. Results The geographical distribution of Blastocystis subtypes is not random, with ST4 almost absent outside Europe. Results Chronic infection with the same subtype Relative abundance (A,B) and breadth of coverage (C,D) of Blastocystis in infected subjects over two time points. The ability to cause chronic infection is not dependent on particular subtypes 11

Results The prevalence of Blastocystis is higher in normal weigth subjects compared with obese ones. A strong correlation with Body Mass Index exist.

Implications: Very common intestinal flagellate of the human gut Only two genoypes (1 and 2) described so far, of which genotype 1 largely predominates in humans, worldwide.

12 Results The prevalence of Blastocystis is higher in normal weigth subjects compared with obese ones. A strong correlation with Body Mass Index exist. Dientamoeba fragilis Our research goal was the identification of genetic markers useful for epidemiologic studies. Indeed, only ribosomal and a few house-keeing genes are available. Implications: Very common intestinal flagellate of the human gut Only two genoypes (1 and 2) described so far, of which genotype 1 largely predominates in humans, worldwide. Clinical relevance unsettled and debated Relation between genotypes and clinical manifestation? Transmission routes largely unknown (zoonotic?) Experimental approach We used genomic DNA from two subjects infected with D. fragilis and sequence total DNA by pyrosequencing. We generated a total of 455,798 reads comprising 200 Mbp (average length, 426 bp) and a total of 320,894 reads comprising 137 Mbp (average length, 457 bp), respectively, form the two specimens. Raw sequences were analysed using a metagenomics analysis server (MG-RAST, that provides taxonomic classification of sequences by interrogating both whole reference genomes and ribosomal databases, and assign sequences to various taxonomic levels based on the observed sequence similarity. Results-1 A very small fraction (< 1%) of the DNA sequences was classified as being of eukaryotic origin. We focused on those having significant similarity to Trichomonas vaginalis and identified a few hundred sequences putatively originating from D. fragilis Loci putatively encoding for a cathepsin L-like cysteine peptidase, a laminin A family protein, a TKL family protein kinase, a peptidase T-like metallo-peptidase, a serine peptidase, and a protein were selected. Loci were validated by nested PCR assays on DNAs extracted from D. fragilis positive stools. Two other markers (18S rdna, and large subunit of RNA polymerase II ) were also included. 12

Results-2 A clonal organism? A total of 111 isolates from symptomatic and asymptomatic persons of different age groups in Italy, Denmark, Brazil and Australia were genotyped.

By sequencing about 500 PCR products (totaling 160 kb of DNA sequences), a very low level of polymorphism was observed across all the investigated loci, suggesting the existence of a major clone of D.

13 Results-2 A clonal organism? A total of 111 isolates from symptomatic and asymptomatic persons of different age groups in Italy, Denmark, Brazil and Australia were genotyped. All, except one, belonged to genotype 1. By sequencing about 500 PCR products (totaling 160 kb of DNA sequences), a very low level of polymorphism was observed across all the investigated loci, suggesting the existence of a major clone of D. fragilis with a widespread geographical distribution. MLG2 (IT) MLG6 (IT, AUS) MLG1 (IT, AUS DK, BR) MLG5 (IT) MLG4 (IT) MLG7 (IT) MLG3 (IT, DK) In short NGS technologies have revolutionized the way (complex) biological questions are addressed Cost is decreasing whilst data generation capacity is increasing Applications are unlimited Speficic challenges exist for parasites (but can be overcome) Parasites are extremely interesting biological models for NGS studies! QUESTIONS? QUESTIONS? 13

14 Sanger DNA sequencing Illumina: principle of sequencing paired ends versus mate pairs 14

Matthew Tinning Australian Genome Research Facility. July 2012

Next-Generation Sequencing: an overview of technologies and applications Matthew Tinning Australian Genome Research Facility July 2012 History of Sequencing Where have we been? 1869 Discovery of DNA 1909