SVM 2FG

VEB GGC

MMI MET

EV EV

GGC

2FG MMI

Sequencing VEB

Libraries Mechanical Enzymatic Shearing of DNA by nebulization. Shearing of DNA by sonication. Enzymatic DpnII (GATC) NlaIII (CATG)

Sanger Sequencing

Clonally amplified Templates Single-molecule Templates Libraries Templates Clonally amplified Templates Single-molecule Templates

Clonally amplified Templates 1) Emulsion PCR

Emulsion PCR Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR 40 to 60 cycles Water-in-oil emulsions (also referred to as “reverse emulsions”) consists of water droplets suspended in oil. In emulsion PCR each of these droplets is referred to as a microreactor an the concentrations of the reactants are set up so that the average microreactor contains less than 1 fragment of target DNA. The presence of surfactants prevents the water droplets from coalescing.. The primers and Polymerase are in excess so every microreactor will contain adequate primer and enzyme, the concentration of target DNA is arranged so that each microreactor will contain <1 target, thus minimizing the number of times that multiple targets are placed on a bead. Microreactor contents Bead target Primers /polymerase result 1 1 in excess single product clonal bead 1 0 in excess No product 2 1 in excess multiple single product clonal beads (each bead lower signal) 1 2 in excess poly clonal bead Note that there are a massive number of microreactors in 1ml of solution > 1010 thus as long as you can enrich for the successful reactions it is not an issue if many microreactors do not “work” Emulsion setup ePCR

Index Lane Max samples 48 * 8 = 384 Index Lane Max samples 3 * 8 = 48 Index Lane Max samples 1 * 8 = 8

Clonally amplified Templates 2) Solid-phase amplification

Single-molecule Templates 1)Primer or template immobilized

Single-molecule Templates 2)Polymerase Immobilized

Clonally amplified Templates Single-molecule Templates Sequencing by ligation Pyrosequencing Cyclic reversible termination Real-time Sequencing

Cyclic reversible termination 1)3’-blocked reversible terminators Modified nucleotides 1)3’-blocked reversible terminators 3’-O-allyl 3’-O-azidomethyl

2)3’-unblocked reversible terminators Modified nucleotides 2)3’-unblocked reversible terminators

Cyclic reversible termination 4 colour

Phasing and pre-phasing are caused by incomplete removal of the 3' terminators and fluorophores, sequences in the cluster missing an incorporation cycle, as well as by the incorporation of nucleotides without effective 3' terminators. Phasing and pre-phasing cause the extracted intensities for a specific cycle to consist of the signal of the current cycle as well as noise from the preceding and following cycles. As the number of cycles increases, the fraction of sequences per cluster affected by phasing increases, hampering the identification of the correct base.

Cyclic reversible termination 1 colour

Sequencing by ligation

Properties of the Probes Cleavage site is between 5th and 6th base 3’ 3’ ligation site, cleavage site and dye are spatially separated Fluorescent dye interrogates base on 1st + 2nd position X X n n n z z z Blue-probe The last three bases are shown as z as they are universal bases and do not play a role in the specificity of the ligase. The ligase used (T4 DNA Ligase) needs a length of 8 nucleotides for ligation (oligos less than this length will not be ligated by T4 ligase). However, there is only fidelity in the first 5 bases (bases must be correct in these positions for the ligase to seal the junction), so the last 3 bases in the sequence need not be correct. Universal bases have no (significant) bias in hybridization potential, so these are used to reduce probe pool complexity, while also contributing to probe stacking/stability. Using this probe design reduces the probe pool complexity to 1 correct sequence per 1024 probes (256/dye) where full degeneracy (7 generate bases and 1 cleavable) would be 1 correct sequence per 16,384 probes). Note in actuality we use a two base encoding system, this is complex and is being omitted from this slide deck as it has to many new concepts to introduce at once. The two base encoding system facilitates identification of sequencing errors as it enables discrimination between an incorrect base call and a SNP when using a reference sequence. Why is the A probe called the A probe when it is actually measuring the presence of a T in the target? As we will end up constructing a 25bp complementary sequence the A probe is the so called as that will be the base present in the finished 25bp complementary sequence. Probes are octamers N=degenerate bases, Z=universal bases 1024 probes, 256 probes per color

SOLiD 4-color ligation Ligation reaction 3’ p5’ universal seq primer ligase Y-probe 5’ 3’ B-probe G-probe R-probe X Xn n n z z z X X n n n z z z Note: Here is the first slide where we point out the fact that ligation is working in the opposite orientation that many people are used to. Standard sequencing is done using polymerases to extend strands in the 5’3’ direction. In the SOLiD system ligated products are built in the 3’ to 5’ direction. [An aside about this technology: The ligation can be done in either direction as the ligase needs only a phosphorylated junction (or ‘nick’). Therefore, ligation-based sequencing can be done using a 5’ phosphorylated sequencing primer (as shown) but it can also be done in the other direction using 5’-phosphorylated probes (and nonphsphorylated sequencing primer).] For clarity only one target is shown, each bead will have many targets (10,000s) per bead which leads to very bright fluorescent signal. 1µm bead Template Sequence 5’ 3’ P1 Primer 1µm bead

SOLiD 4-color ligation Ligation reaction ligase Y-probe 5’ 3’ B-probe G-probe R-probe X Xn n n z z z X X n n n z z z ligase x x After ligation, there is a capping step (with phosphatase) which will remove the 5’ phosphate from any un-extended primer. Removing the 5’phosphate from strands not extended in the previous sequencing round will render them inactive in the next ligation reaction. This capping step therefore reduces dephasing of template strands. universal seq primer 1µm bead p5’ Template Sequence 5’ 3’ P1 Primer 1µm bead

SOLiD 4-color ligation Visualization x x Wash out unligated probes, and image array using a powerful xenon light source (no lasers). Each bead will light up in 1 of 4 colors (color corresponds to either A, C, G or T). Bead images are recorded with the color of each defined at each cycle. universal seq primer 1µm bead Template Sequence 5’ 3’ P1 Primer 1µm bead Y 1-2

SOLiD 4-color ligation Cleavage p5’ x x Probes are cleaved in an efficient chemical reaction and occur between the 5th and 6th base. This generates a 5’ phosphate that is essential for the next cycle of ligation to occur. Any primer (or DNA end) that did not ligate had its 5’ phosphate removed earlier during the phosphatase step. Only strands with a 5’ phosphate can participate in the next round of ligation. universal seq primer 1µm bead Template Sequence 5’ 3’ P1 Primer 1µm bead Y 1-2

SOLiD 4-color ligation Ligation (2nd cycle) ligase Y-probe 5’ 3’ B-probe G-probe R-probe X Xn n n z z z X X n n n z z z x x ligase Note that as first ligation probe has been cleaved it is now 5 base pairs long so second base queried will be at the 10th position universal seq primer 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead x x Y 1-2

SOLiD 4-color ligation Visualization (2nd cycle) x x Read second position (10th base) universal seq primer 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead X X Y 1-2 R 6-7

SOLiD 4-color ligation Cleavage (2nd cycle) p5’ x x Cleavage occurs between 5th and 6th base. It generates a 5’ phosphate (any primer that did not ligate had its 5’ phosphate removed earlier , the net effect is that it is blocked from future ligation, thus preventing dephasing) universal seq primer 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead X X Y 1-2 R 6-7

SOLiD 4-color ligation interrogates every 4th-5th base Note when giving this presentation after the 5th probe (25) shows up you want to start discussing the reset reaction as the next click will reveal the cleaned up target Note the final probe does not need to be cleaved as it will be removed at reset. universal seq primer 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead X X X X X X X X X X Y 1-2 R 6-7 R 11-12 B 16-17 G 21-22

SOLiD 4-color ligation Reset This is the result of a reset: the extended template strand is melted off and the sequencing template re-exposed as a fresh, clean template and devoid of any noise generated by previous sequencing cycles. In this method, noise is generated by the attrition of strands at each cycle (uncompleted extensions) that serve to reduce template number (and therefore, fluorescence intensity) on each bead, and increases the noise-to-signal ratio of each bead. The ability to ‘reset’ is one of the major benefits of this chemistry. A major problem that limits read length of all NGS systems is that as the read gets longer, the signal falls and noise rises, until you can no longer accurately call a base. By resetting the system every 5 cycles we remove all the accumulated noise at each sequencing round. 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead

SOLiD 4-color ligation (1st cycle after reset) 3’ p5’ universal seq primer n-1 ligase Y-probe 5’ 3’ B-probe G-probe R-probe X Xn n n z z z X X n n n z z z x x ligase The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing. universal seq primer n-1 1µm bead p5’ Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead

SOLiD 4-color ligation (1st cycle after reset) x x The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing. universal seq primer n-1 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead R 0-1

SOLiD 4-color ligation (2nd Round) The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing. universal seq primer n-1 1µm bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead X X R 0-1 R 5-6 10-11 G 20-21 B 15-16

Sequential rounds of sequencing Multiple cycles per round bead Template Sequence 5’ 3’ Adapter Oligo Sequence 1µm bead 3’ universal seq primer 1-2 6-7 11-12 16-17 21-22 reset universal seq primer n-1 15-16 20-21 3’ 0-1 5-6 10-11 reset universal seq primer n+3 spacer 3’ 4-5 9-10 14-15 19-20 24-25 Again the reset is one of the unique attributes of Ligation based sequencing, A first round and four resets give a total of 5 rounds of sequencing that records the color at every 5th position to generate a 25 base read length. Feasibility out to 50 bases has been demonstrated (5 rounds of 10 cycles). reset universal seq primer n+2 spacer 3’ 3-4 8-9 13-14 18-19 23-24 reset universal seq primer n+1 spacer 3’ 2-3 7-8 12-13 17-18 22-23

2 Base Pair Encoding Using 4 Dyes Red-probe 5’ A T n n n z z z 3’ A C G T 2nd Base 1st Base Blue-probe 5’ T T n n n z z z 3’ On our probes the 1st base encoded is position 4 the 2nd base encoded is position 5 What does two base encoding mean? this chart shows the color seen when a specific pair of bases is interrogated 1st base refers to the first of the pair of bases (sometimes referred to as the leading base) and the 2nd base to the 5th base in the probe (sometimes referred to as the trailing base) We are analyzing the 4th and 5th base the chart shows the color assigned if the 4th base is T and 5th base is T a blue signal is seen if the 4th base is A and 5th is a T then red will be seen Note as we transition to AB dyes the color scheme will change (the colors are recorded as 0,1,2,3)

Advantages of 2 base pair encoding Each base is interrogated twice, by two independent probes A C G G T C G T C G T G T G C G T A C G G T C G T C G T G T G C G T Just to emphasize each base in the sequence is interrogated twice, hopefully with the same result

Pyrosequencing

Solid

Solid 485M F3 reads 485M R3 reads 8-9x Hg19

Solid Barcode reads Reject reads 30M F3 reads 30M R3 reads

Solid

Solid

Solid

Solid

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina It provides a simple extension to the FASTA format: the ability to store a numeric quality score associated with each nucleotide in a sequence.

FASTQ from SFF FASTQ from HiSeq2000 S_1_1 2.7G S_1_2 2.7G

FASTQ S1_1 + S1_2 SAM 7.4G 2.7G + 2.7G

A BAM file (.bam) is the binary version of a SAM file. FastaQ 5.7G SAM 7.4G BAM 2.1G