BIOL 325 GENOME/EXOME/RNA SEQUENCING STUDY GUIDE

explain how genetic information is stored within the double helix (written response question)

Genetic information is stored within the double helix through the specific sequences of the four nucleotide bases that complementary pair together. Adenine goes with Thymine and Cytosine goes with Guanine. The specific sequences that these pairs form encode the genetic instructions for building and maintaining an organism

what does every sequencing method/process needed?

• needs a labeled primer

  • primer needs to hit right before the sequence of interest to prime and start the sequencing process

• needs high quality template

  • isolated DNA or RNA

• needs dideoxynucleotides

  • dATP, dCTP, dGTP, dTTP

  • added separately to each of the four tubes

what is sanger sequencing?

DNA replication in a test tube that terminates a growing chain through dideoxynucleotides

what are dideoxynucleotides? how does the removal of the -OH group impact the addition of nucleotides?

• dideoxynucleotides remove one of the hydroxyl (-OH) groups to not allow a phosphodiester bond to form which in turn terminates the growing chain

• the removal of an -OH does not allow to chain to continue because the next nucleotide needs the -OH

what is the purpose of adding an enzyme into the sanger (chain terminating) sequence method?

with addition of enzyme (DNA polymerase), the primer is extended until a dideoxynucleotide (ddNTP) is encountered which will end the chain

how is ratio important in sanger sequencing?

with the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template

how do you set up chain termination? what would be an example of it?

• in four different tubes they will have the DNA polymerase, a mix of dideoxynucleotides and standard nucleotides

  • ex. for the adenine tube —> 4 dNTPs + 1 ddATP = would want more normal ATP than the dideoxy so some normal will get incorporated into the sequence and eventually get an A but when the ddATP gets added = chain ends at ddA

• all chain will end with whatever dideoxy was added

  • ddCTP = ddC

  • ddGTP = ddG

  • ddTTP = ddT

name and describe the standard method for the determination of nucleotide sequences (written response question)

The standard method for determination of nucleotide sequences is the sanger sequencing method. The sanger sequencing method is a chain termination sequence where a DNA polymerase or an enzyme will extend the primer until a specific dideoxy is encountered which will terminate the sequence of the specific dideoxynucleotide. The ratio of the dideoxynucleotides and the standard nucleotides is crucial in the Sanger sequencing method and each dideoxynucleotide is labeled with a fluorescent dye for specific identification of each of the nucleotide bases. By resolving the terminated chains through electrophoresis, a sequencing ladder occurs where an individual can determine the sequence by reading the gel from bottom to top/smallest fragment to largest fragment.

what is cycle sequencing?

• is a chain termination sequencing done in a thermal cycler

  • done in a thermal cycler to cycle through the replication process thousands of times to hit every NT in that specific tube

• it is amplification based son heat-stable DNA polymerase is needed because if a non heat-stable polymerase is used, it is killed during the denaturing step

what are fluorescent dyes? what is fluorescently tagged in sequencing methods?

• are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths

• the primer OR nucleotides are fluorescently tagged and could be tagged with hundreds of different types of dyes

what are fluorescent dyes based on? what is covalently attached to the molecules?

• a lot of fluorescent tags/dyes are based on fluorescein (green) or rhodamine (red) derivatives

• for sequencing applications, these dyes/molecules can be covalently attached to molecules

what is dye primer sequencing?

primer is tagged with fluorescent due conjugated nucleotides at the 5’ end of the chain

• the 5’ end that was sequenced will be fluorescent s the size based output will tell what the end nucleotide is

what is dye terminator sequencing? what is needed for each of the four dideoxynucleotides?

• fluorescent dye molecules are covalently attached to the dideoxynucleotide at the 3’ end of the chains

• since nucleotide is tagged —> need four color for the different nucleotides to detect them

  • an instrument will detect all the different emissions which will help detect all the four nucleotides that will have distinct dyes/colors

• all four reactions can be dine in a single tube because of the different colors used

what is the DNA ladder for DNA terminator sequencing read on?

electropherogram

what is automated sequencing?

• uses a gel capillary tube with an instrument/electropherogram to detect different emissions

• dye primer or dye terminator sequencing on capillary instruments

• sequence analysis software provides analyzed sequences in text and electropherogram form

• peak patterns reflect mutations or sequence changes

understand how to read data from an electropherogram (written response question)

To read data from an electropherogram, the different nucleotides will have different peaks and colors that are specific to the nucleotide bases. The heights of the nucleotide peaks will not always be perfect compared to each other however they should be fairly close. no peak for a nucleotide means that there was an error in the read. The order of the nucleotides can be read through their different colors and heights which will help in determining the sequence but also determine if there is a single mutation present.

what is a homozygous mutation?

• T/T or A/A mutation = one allele is mutated and the other stays normal and happens quite often in mutations especially cancer

• can tell because a peak of a T or an A is similar to the other peak of the same nucleotide = both alleles have a T or an A

what is a heterozygous mutation?

mutation that has another peak of a different nucleotide in another nucleotide

what would be considered good sequence data? what would be considered an ugly piece of data and what would be a bad read?

• both strands are sequenced and should both give a complementary output

• sometimes the peaks are not perfect so that is why the other strand is sequenced to confirm what nucleotide the non-perfect peak is

• sometimes there are compressions where the nucleotide is pushed up against a secondary structure in the DNA

  • like it is looped out or loop in and it cannot be read meaning that it is an ugly piece of data

• if the other strand looks good and the other strands nucleotide is not clear but can be inferred then you can trust the sequencing data

• if there are values or peaks on both of them at the same location then it is a bad read so you cannot trust the sequence data

what are other instances of bad sequence data and what would their solution be?

• problem = overloaded/too much DNA

  • solution = check concentration of template

• problem = dirty sample —> protein or lipid contamination

  • solution = clean up DNA/use DNA clean up kits

• problem = too much voltage applied to the capillary gel/slowly gets higher and higher throughout the assay

  • solution = check voltage on the machine

• problem = short reads (more common and usually the ratio of the nucleotides are messed up)

  • solution = too much ddNTPS to dNTPs or low processivity enzyme so fix ratio/add more processivity enzyme

  • too much DNA or too little DNA in comparison to nucleotide ratio —> will not read will

what is RAS mutant? how was an electropherogram used to sequence and detect RAS mutant? how can you confirm the mutant?

• RAS mutant = oncogene/mutated gene that can cause cancer and commonly mutated in LOTS of cancers

• in a normal codon sequence, there can be two peaks that are the same height if they are the same nucleotide but they are still unique peaks

• electropherogram detects the heterozygous mutation in the RAS gene of G>T in exon 12

• can confirm them mutation by reading the other strand because the other strand will have similar peaks in the same location as the other strand

what is BRCA1? how was an electropherogram used to sequence and detect BRCA1 mutation?

• BRCA1 = breast cancer susceptibility gene

  • supposed to help control the cells but once mutated, the cells grow uncontrolled

  • many mutations in the BRCA1 gene but top 10 is deletion of two nucleotides, A and G, next to each other (frameshift mutation)

• electrophreogram detects the frameshift of the BRCA1 mutation and the peaks will start getting messed up and the peaks will begin to go inside each other and there will be gaps

• cheap assay/test

define and describe the purpose of bisulfite sequencing (written response question)

Bisulfite sequencing is used to detect methylation in DNA. Bisulfite deaminates/removes amine group from cytosine making uracil. The methylated cytosine stays as cytosine, while the unmethylated cytosine turns into uracil. A methylated cytosine is NOT changed by bisulfite treatment. The bisulfite-treated template is then sequenced and a highly methylated sequence is a sign of the region being silenced.

what is high-throughput or next gen sequencing? what does it apply to?

• high demand resulted in low cost and fast methodologies

• high-throughput sequencing includes next gen “short-read” and third generation “long-read” sequencing method which applies to

  • genome sequencing

  • exome sequencing

  • transcriptome profiling (RNA sequence)

  • DNA-protein interactions (ChIP-sequence)

  • epigenome characterization

what are the five sequencing approaches for next gen sequencing?

• pyrosequencing (2nd gen)

• ion cunductance

• reversible terminator

• ligation mediated (SOLID)

• real time or phospholinked nucleotides

what is pyrosequencing?

• when a nucleotide is added to the growing reaction, light is emitted and can determine what nucleotide was added by detecting the light

• each nucleotide is added one at a time and only one of the four will generate a light signal and it is washed away and repeated again

• light signal is recorded on a pyrogram

what is the process for pyrosequencing?

• only one out of the three phosphate groups in the added nucleotide is needed to hook it to the next nucleotide so the other two phosphate groups are released (PPi is removed)

• experiment part = use an enzyme with a substrate that turns the diphosphate into ATP

• when the substrate luciferin in the enzyme luciferase, it will break down ATP and produce light

  • detector needed to detect light

what is ion semi conductor sequencing?

• whenever a nucleotide is added, a proton (H+) gets released

• based on the detection of hydrogen ions released during DNA polymerization

• H+ released changed the pH of the solution which is detected by an ISFET

  • the unattached dNTP molecules are washed out before the next cycle

how is the signal detected in ion semi conductor sequencing?

• start with micro wells (96 well plate) and when the nucleotide is added, a proton (H+) is released

• the well is sitting on a ion sensitive layer so when the ion is released, it gets excited

• below the ion sensitive later is an ISFET ion sensor that reads the excitement and creates a signal to the computer

• all are contained within a semiconductor chip which is similar to what is used in the electronics industry

what are the pros and cons for ion semi conductor sequencing?

PROS

• rapid sequencing speed

• low operating costs

  • only sensor is needed

CONS

• short read length

what is ion semi conductor sequencing best suited for?

• small applications like

  • nucleic acid mutations

  • microbial genome

  • transcriptome sequencing

  • targeted sequencing

what is illumina dye sequencing aka bridge amplification or reversible dye termination?

• automated, simple and efficient

• can sequence multiple strands at once = longer reads

• each NT is labeled with a different fluor

what is the summarized version of the process of illumina dye sequencing?

• adapters are added to the ends of the DNA fragments and through reduced cycle amplification, additional motifs are introduced like sequencing binding site, indices, and regions complementary to the flow cell oligos

  • the oligos are complementary to the adapters on the DNA fragments

• illumina dye sequencing produces clusters

• basically one strand is read then it recreates the second strand so the second strand is sequenced

  • both strands need to be sequenced to confirm if you have a correct sequence

what are the uses for illumina dye sequencing?

• uses reversible dye-terminators which enables the ID of single nucleotides as they are washed over DNA strands

• used for

  • whole genome sequencing (WGS)

  • regional

  • transcriptome

  • metagenomics

  • small RNA discovery

  • methylation profiling

  • protein-nucleic acid interaction analysis

name two technological updates that have greatly improved DNA sequencing (written response question)

one technological update that has greatly improved DNA sequencing is pyrosequencing. pyrosequencing provided a bright way to determine a DNA sequence by emitting light whenever a certain nucleotide was introduced. another technological update that has greatly improved DNA sequencing is illumina dye sequencing. illumina dye sequencing allows multiple strands to be sequenced at once and is automated.

how has NextGen sequencing revolutionzed the process? distinguish and describe these subtypes: pyrosequencing, ion semiconductor, illumina dye (written response question)

NextGen sequencing has revolutionized the process as it now allows for high-throughput sequencing, which allows the process to be low cost and faster. Pyrosequencing is a second-generation sequencing method where a nucleotide emits light as it is added to the growing reaction. Ion semiconductor method is where, instead of light, a proton is released when a nucleotide is added. Illumina dye sequencing is when fragmented DNA is attached through a flow cell and forms a bridge to another adapter, and is amplified to form clusters. Each nucleotide on the forward and reverse strands is read through reversible dyes and should be complementary to each other.

what is genomics?

• attempts to study entire genomes and their organizations

• provides contextual framework for genome information

what was the first genome sequenced in 1984?

EBV (epstein barr virus)

what is human genome project (NIH)?

• started with known sequences and used the known sequence to walk and prime into unknown sequences

• published in 2000 a week apart from celera but incomplete

what is ventner’s group, celera?

• ventner sequenced his own genome

• whole genome shotgun which cut genome into fragments and sequenced —> used computer to overlap the fragments

  • assembly is HARD, often leaves gaps

• published in 2000 a week apart from HGP but incomplete

how much did the first genome cost at first, in 2006, and 2014?

• first human genome sequenced = $2 billion

• 2006 = $50 million

• 2014 = $1,000

what is the 1000 genomes project from 2008?

• sequenced 1000 genomes to identify variations in the human genome

  • national human genome research institute (NHGRI), wellcome trust sanger institute, and the beijing genomics institute

what is workflow of whole genome shotgun sequencing (WGSS)?

• entire genome is sheared randomly into small fragments by enzymes, sonicator, or mechanically

  • how long the genome is sheared will determine how big the fragments will end up being

• the reasonable fragment sizes are primed off of and sequenced

• a computer is used to realign/overlap the sequenced fragments but it is slow

what is workflow of hierarchical shotgun sequencing?

• genome is broken into thirds then further sheared into reasonable fragment sizes and their order is deducted

  • the further sheared DNA fragments is sequenced and overlapped to construct the genome consensus

how are entire genomes sequenced today? (written response question)

Entire genomes are sequenced through whole genome shotgun sequencing. Whole genome shotgun sequencing shears an entire genome and the reasonable fragments are sequenced and primed off of. The sequenced fragments are then overlapped using a computer to construct the genome.

what are the mutants to wild type comparisons?

• cancer genetics

  • take area of patients tumor that seems to be behaving differently than another area so the entire genome of each of them is sequenced and see how they compare to each other

• antibiotics or drug resistance

• targeted therapies

what is ligation sequencing (ABI SOlID)?

sequence method that reads two nucleotides at a time

what is the process for ligation sequencing?

• magnetic bead(s) is created with an adapter sequence on it and primers complementary to the adapter sequence

• dye nucleotide sequences are labeled

  • every combination of a nucleotide sequence is on the probe, and each has a different color in it

• add a big mix of probes and let them adhere to whatever the correct complementary sequence is

  • needs LIGASE to hold the probes to the sequence

• ligase adds the probe to the nucleotides and gives it color

  • now know what the two nucleotides are

• wash everything away and add probes back to the sequence to find the next two nucleotides

what is the NGS workflow for nucleic acid extraction?

• isolate nucleotides first and it could be from any sample like solid tissue or fluid/plasma

• use kit to extract NA from sample

• do quality control to make sure it is clean and not degraded

what is the NGS workflow for library prep?

• ligate adapter on the DNA

• for genome sequencing, size select the DNA that was sheared

  • probably will use spin column

• amplification/prep

• quality control

what is the NGS workflow for sequencing and analysis?

• sequence

• data analysis

  • base calling

  • read alignment

  • variant calling

  • variant annotation

• human will look at mutation location and loading it into other data bases

what is DNA barcoding? how does this relate to when a new outbreak occurs?

• it is when a sequence that is unique to an organism is scanned and created into a unique ID/barcode

  • the genome that is unique to the organism tends to be the mitochondrial gene which makes cox genes

• if a new outbreak occurs, the organism is sequenced and compared to barcodes of known organisms to have an idea to what is causes the new outbreak

name/describe the three ways to prepare a template for sequencing (written response question)

1. emulsion PCR: amplifies DNA in small water-in-oil droplets while keeping similar sequences apart to avoid formation of artifacts

2. rolling circle PCR: amplifies the template by adding complementary adapters onto sheared DNA, making them overlap each other and creating a circle. A polymerase is added to make a really long repetitive copy of that DNA fragment and is primed and sequenced.

3. solid-phase amplification: amplifies the template by forming bridges on the flow cell by complementary binding to adapters and forming clusters of identical strands

what are the run times of NGS, their read lengths, and their error rates?

• run times range from 10 hrs to 5 days

• read lengths are 35-400 bases

• error rates are worse but the output in gigabases are larger

what is massive parallel sequencing analysis?

• input samples must be a library of random amplified DNA or sequences attaches to adapter sequences

• each fragment is amplified on a solid sequence — each cluster then is one set of sequencing reactions

since humans share 99.9% of their genome, what is the 0.1%? what is the cause of 80% of rare disease?

• 0.1% of genes trigger the difference in our development

• over 80% of rare disease are caused by genetic mutations in that tiny difference, affect ~8% of the population (1 out of the 1000 disease)

  • detecting such diseases is challenging

what does NGS not require for genome sequences?

does not require a capture step for genome sequences which offer coverage across the entire genome

what is the exome and what is exome sequencing?

• exome = expressed part of your DNA

• exome sequencing = capture-based method, targets coding regions of the genome

what is the transcriptome and what is transcriptome sequencing?

• transcriptome = the sun of all the mRNA expressed from the genes of an organism

• transcriptome sequencing = capture-based method, detects coding plus multiple forms of RNA

how is RNA sequenced? (written response question)

Before, RNA used to be sequenced by converting the mRNA into cDNA through reverse transcriptase. By converting the mRNA to cDNA, it can cause errors in two areas, making it error prone. Now, RNA is sequenced through the ion-proton system to detect proton that is released. It directly sequences RNA to remove an error-prone step. It has the potential to find cancer mutation that affect intron removal and to compare affected person to normal transcriptome.

what is bioinformatics?

• biology working with pure science, technology, and statistics and work in silico (computer)

• created barcoding database

what are the 6 consensuses sequence data comparisons?

• tumor genomes

• evolution

  • creating phylogenetic tree

• species ID

• environmental response

• putative protein structure

• epeigenetics data

what are computers required to do?

align the sequences

• identity: identical residues (DNA, RNA, or AA)

• similarity: results in AA with same property

• homology: a numerical value of how conserved a sequence is

what is BLAST?

• basic local alignment search tool

• blasts and searches for things

• NIH funded website

  • put info into website and it spits out all the possibilities

• anything that has been sequenced by a funded research lab puts their sequences in BLAST