BLD 430 Exam 3

Describe the workflow of automated dideoxy dye terminator (Sanger) sequencing

1. amplify target via PCR, confirm amplification, purify 2. prepare and run cycle sequencing on thermocycler. Set up is similar to PCR 3. remove excess dye terminators via ethanol precipitation or spin/gel/bead columns 4. fragments are fully denatured before running in gel or capillary. Heat sequencing ladder (product) to 95-98C to denature and snap cool on ice to maintain single stranded confirmation. Denaturing conditions maintained so fragments are resolved by size. Formamide, 50-60C 5. detection. Fragments pass thru clear capillary laser. Excited fluorochrome labeled ddNTP fragments. Fluorescence is emitted and captured by detector. Detector converts fluorescence to electric signal. Processed in software as red, blue, black, or green peak in electropherogam

Analyze a RFLP gel and southern blot when used for human identification and parentage testing

1st tool used? ABO blood group antigens, but low discriminating power because only have 4 groups, only able to exclude. HLA typing added higher statistical power because of increased polymorphism. Testing both did not provide positive identification though. Tandem repeat sequences (insertion or deletion repeats), loss or gain of one repeat can be resolved using electrophoresis. Digesting human DNA with HaeIII, fragments small enough to resolve on gel with informative pattern.

Compare and contrast interphase and metaphase FISH

Advantages of interphase FISH: can use fixed cells, no need to culture to metaphase (faster TAT), increased sensitivity due to evaluation of a hundred cells (compared to 20 cells with metaphase procedures) Disadvantages of interphase FISH: limited to detection of genetic changed specific to probes (karyotyping can detect any chromosomal change (with varying sensitivity))

compare the phenotypic consequences of different types of point mutations

All mutations have the potential to effect protein sequence (large differences can lead to significant impact. Silent point mutations do not effect the amino acid sequence. Conservative is a change in the amino acid sequence with another amino acid that has similar biochemical properties. Non-conservative is a change in amino acid sequence with another amino acid that is significantly different than the wild type. Nonsense is a mutation that terminates protein prematurely by introducing a stop codon into the reading frame at the ribosome. A frameshift mutation is an insertion or deletion of non-multiple of 3 nucleotides.

Discuss targeted NGS libraries; compare and contrast the two primary methods of enrichment.

Amplicon-based enrichment is versatile and easy to use method. Need to consider primer design because target sequence variability can lower primer to target binding efficiency or non amplification leads to allele drop out, with inaccurate assessment of variant allele frequencies. Use overlapping primers to reduce dropout. Both probe an amplicon enrichment methods are affected by GC rich target regions. Have secondary structure, lowers binding of probes or primers. GC rich regions "clamp" primers in amplicon enrichment, lower PCR efficiency. AT regions can have poor probe hybridization, loss of sequencing template fragments

Provide a broad description of molecular antibiotic susceptibility and epidemiologic testing

Antibiotic resistance: susceptibility and minimum inhibitory concentration testing is standard. There is a genetic component to resistance. Mutation resulting in survival or growth advantage in the presence of low-level antibiotics. Acquisition of genetic resistance factors vis conjugation, transposon. Epidemiologic testing assesses an outbreak, small or widespread in healthcare, food, community settings, pandemic, etc. Characterize genetic factors that help to identify and mitigate infectious disease spread.

Discuss technical items to consider with STR genotyping analysis including artifacts, gel/capillary precision and resolution, stutter, and binning

Band migration sources of interference: air bubbles, crystals, dye blobs, sample contaminants, temp variations, voltage spikes, etc. Extra bands can be present due to non-template addition of 3' adenine residue. Stutter: artifact of PCR amplification process, where polymerase misses adding a repeat during amplification, this results in different bands/peaks of amplified product. Binning/locus specific brackets: identify where loci and their alleles migrate.

Discuss bioinformatics and how it is used to analyze NGS sequence

Bioinformatics use technology as tool to store, organize, and index large amounts of biological information and complete analysis in silico (by computer) and computational biology. Need standard expression of sequence data, have IUB universal nomenclature. Consensus sequences are sequences with proportional representation of polymorphic sequences. Can also compare sequence info to large regions of DNA in database. Basic Local Alignment Search Tool (BLAST) - nucleic acid and protein level sequence identification to known sample, adjust parameters, compare species

Describe the various data that are used for annotation of NGS sequences.

Confidence, coverage, filtering. Effects on protein can be estimated using algorithms - PolyPhen, SIFT

Distinguish the detection of known mutations from scanning for unknown mutations

Disease related to genotype Factor V, hereditary hemochromatosis C282Y, H63D, and S65C have mutations that occur at same genetic location Some diseases are caused by one or more of many potential mutations Cystic fibrosis transmembrane regulator-600 mutations BRAC1 and BRAC2 genes >2,000 cancer susceptibility mutations Some disease-causing genes are still unknown Need to choose between sequencing or biochemical methods to detect protein alteration Biochemical methods analyze protein structure, may be more appropriate in some cases Enzyme immunoassays, high performance liquid chromatography, mass spec

Describe the procedure for interphase FISH

Do not need to culture cells Evaluation prenatal samples, tumors, hematologic malignancies (not easily brought to metaphase in culture) Fixed cells are exposed to 60-200kb fragment of DNA covalently bound to fluorescent molecule. Probe is complementary to target. For probe binding to autosome, yield 2 signals (maternal and paternal). Deletion or duplication will result in change in signal. Translocation will change signal (separation) Protocol: 1. optimal sample? fresh interphase cells, but can use cells from fresh frozen paraffin embedded tissue 2. treat cells with protease to remove interference from cytoplasmic proteins, fix if not already done, deparaffinize if needed 3. denature probe and target 4. run hybridization at 37-42C in humidified chamber 5. rinse away unbound probe, evaluate under fluorescent microscope for signal. Need adequate numbers of cells. Need pos and neg control. Be aware of photo bleaching of probe.

Discuss NGS gene panels, how many gene targets are typically included in clinical panels, and provide examples of gene panels used for NGS.

Gene panels: primer or probe sets designed to amplify specific genes, regions, or entire exomes (protein coding sequence). Also can construct sets to compare sequences of many organisms or multiple base position differences in highly polymorphic gene (CTFR, HLA genes). Few to >1000 target genes, most clinical panels are 15-500 genes. Target actionable variants that would benefit from therapeutic intervention (cardiomyopathies, muscular dystrophy, cancer (hematologic like lymphoid and myeloid and solid tumor like lung, breast, colon, etc)) Run whole genome for gene discovery? Use >3000 targets

Describe the use of STR genotyping in human identification, parentage testing, and the amelogenin locus for sex identification.

Genotyping: STR typing results in bands or peaks that need to be converted to genotype: allele identification. STR locus allele is defined by its number of repeats. Can be heterozygous or homozygous. Gender identification: amelogenin locus analyzed with STR. Not STR locus, but located on the X and Y chromosomes. Y allele is 6 bp larger than X allele. Evaluation allows for sex determination.

Discuss characteristics of good and poor quality Sanger sequence electropherograms

Good quality: clean baseline, strong, even signal, with one color at each position Poor quality: has uneven baseline, varying peak intensity, more than one peak per position. Dye blobs.

Explain the concept of separation and detection by HPLC, chromatography, and spectrometry

HPLC: Characterizes amino acids via migration patterns Chromatography: Sample is introduced to column and vaporized into a gas Effluent monitored by detector Often coupled with mass spectrometry Mass spectrometry: Molecules are converted to ions that move in magnetic fields based on the mass and charge to create protein signatures Nucleic acids can be analyzed by this by using 5' extension and ddNTPs. The mass of the extended primer indicates sequence/ variant at template site

Discuss the clinical significance of viral sub-type identification in HPV and HCV and its clinical impact.

HPV: many types alpha group contains 64 types that infect anogenital mucosal epithelial cells (high risk types are 16, 18, 31, 35, 39, 45, 51, etc). Need to differentiate high risk types (cause cancer) in conjunction with cytology that is equivocal or ambiguous (atypical). Facilitates detection of high risk patients, and increased monitoring of cervical cancer development. HCV: RNA virus that causes acute and chronic hepatitis, hepatocellular carcinoma. Genotyping of 6 genotypes informs therapy.

Discuss the advantages of dye terminator sequencing compared to radioactive labeled Sanger sequencing.

Historically was a manual method known as radioactive labeling. ????

Explain the principles of ion-conductance and reversible dye terminator NGS platforms

Ion-conductance: indexed libraries amplified using primers immobilized on beads, adapters on library fragments bind complementarily to immobilized primers. Beads with amplicons on surface placed on solid surface (chip), settle into individual wells. Fragments subject to monitored addition of nucleotides. If nucleotides is complementary and binds, have release of hydrogen ion, changing the pH of solution. Changes in pH of well recorded by software, relates nucleotide sequence. Reaction occurs 100s-1000s of times, produces sequence from million of panel library fragments. Reversible dye terminator: captured or amplified fragments are hybridized to immobilized primers on flow cell, amplified by branch PCR into polonies (collection of PCR products). Polonies are sequenced by sequential addition of fluorescently labeled nucleotides, each one specific for particular fluorochrome color. Image is taken after addition of each nucleotide and the software records color and location. Dye is removed, cycle repeats. This occurs on thousands of polonies simultaneously

Describe how karyotypes are made and state the normal karyotype designations for males and females.

Karyotype is the complete set of chromosomes in the cell. Karyotyping is the direct observation of metaphase chromosomes according to size. Cell collected, grown in culture, stimulated into cell division by mitogen, growth is halted by metaphase, arranged into organized display. Females: XX. Males: XY

Define library as it relates to NGS and discuss the steps of library prep.

Library: sets of 100-500bp size fragments representing the region to be sequenced (whole genome or specific gene regions) step 1 in NGS - create the sequencing library Sequencing library: collection of DNA fragments to be sequenced. Use fragments < 1000 bases long. Genomic DNA is fragmented by various methods, will produced different size fragments: shearing with high frequency acoustic energy, sonication, nebulization, enzymatic treatment (will produce 5' and 3' overhangs that will need to be filled to bind synthetic adaptors) step 2: end repair and adapter ligation End-repair: fragments with 5' or 3' overhangs will be removed or filled in by nuclease or polymerase treatment. Adapter ligation to fragments: single, short dsDNA pieces carrying sequences complementary to a single primer pair. Can also contain sequences that identify sample, this is called indexing or barcoding, allows analysis of many samples in the same reaction, software constructs sequences of fragments with same barcode. Reagent sets are available for library are available for library prep. In clinical work, gene panels are targeted towards disease-specific, clinically relevant genes. Enrich DNA sample for these target using 1 of 2 techniques: probe or primer enrichment. step 3: probe enrichment via hybridization. Hybridize biotinylated probes to specific gene regions (captures sequences in and around region of interest), targeted fragments to be sequenced are selected by hybridization with biotinylated probe which is captured with streptavidiin coated beads. Amplicon enrichment: targeted libraries are selected by multiplex PCR with gene specific primer. 1) gene specific primers are tailed with secondary sequences that index/barcode specific samples from multiple samples in same reaction. 2) indexing primers also have adapter sequence used to anchor fragment to sequencing platform. step 4: sequence! most frequent clinical methods: ion conductance, reversible dye terminator.

List the components and molecular reactions that occur in dideoxy chain termination (Sanger) sequencing.

Modifications of DNA amplification process. Primer binds template, complementary to sequence, 5' of target. Dye terminator method is most common. Deoxynucleotides (dNTPs) and labeled dideoxynucleotides (ddNTP) added to primer by polymerase. Growing chain is terminated upon random addition of labeled ddNTP. Missing hydroxyl group at 3' sugar means that it can't extend phosphate backbone. dNTPs and labeled ddNTPs are randomly incorporated into growing chain during elongation.

Discuss the objectives, strategies and outcomes of the Human Genome project.

Objective: uncover the molecular basis of disease. Genomes of clinically important organisms were also discovered, advanced typing and predicating infectious disease treatment outcomes. Project began by sequencing Epstein-Barr virus (80 genes) in 1985 using manual sequencing, controversial due to cost and risk of only finding non-coding DNA. Capillary electrophoresis, increased resolution and efficiency of project. NIH endorsed Human Genome Project. Goal: to complete 20 Mb of sequence by 2005, BLAST and GRAIL introduced. DNA donated by 100 anonymous volunteers, only 10 genomes sequenced 5-10 times in each region. A parallel project started in other research group, institute for genomic research, with same goal. Goal of NIH project moved to 2003. Competing groups approached problem differently NIH: hierarchical shotgun sequencing, start in areas of known sequence and walk out Celera: whole genome shotgun sequencing, used 10 equivalents of human genome cut into small fragments and sequenced, computers would find overlapping sequences and assemble into proper chromosome locations. Controversial at first due to technical issues and data use. NIH eventually adopted this method. Information from project has lead to evolution of clinical molecular diagnostics, allowed for identification of multicomponent disease. Overall, benefits may have not yielded the benefits to human health that were expected, but provided insight to genes and regulation.

Describe the principles of pyrosequencing and bisulfite sequencing methods and their interpretation.

Pyrosequencing: determine sequence without producing ladder. When dNTP is added complementary to template and added to growing strand, energy is released in the form of pyrophosphate and generates luminescent signal. Process repeated for the 4 bases. Signal detected and complied into a pyrogram. Useful for known short sequence analysis and confirmation typings in mutation detection and infectious disease. Bisulfite sequencing: designed to detect methylated cytosines. This provides information of gene expression, oncology applications. Genomic DNA is cut with restriction enzymes, but should not cut region to be sequences. Run fragments on gel, purify fragments of interest. Denature then incubate with bisulfite solution 16-20 hours. Cytosines will deaminate and convert uracils. 5' methylcytosines are not modified which indicates levels of methylation. Run PCR, compare where cytosines have not converted to T (uracil) compared to untreated sequence. Compare untreated and treated sequences to determine relative number of C to T methylation

Define restriction fragment length polymorphism (RFLP) and discuss how RFLPs are used in genetic mapping, parentage testing, and human identification.

Restriction fragment length polymorphisms (RFLPs): one or more nucleotide changes that alters DNA target of restriction enzyme RFLP typing methods were original DNA target methods used for gene mapping, human identification, and parentage testing. Characteristic differences in DNA fragment sizes generated from restriction enzyme digestion. Fragment sizes vary based on base sequences in target restriction sites. Nucleotide sequence changes may remove, change or create new enzyme cutting sites, alter number of fragments. Parentage testing: have diploid chromosomes and alleles, one from each parent? Use unique combination of alleles to infer parental contribution to offspring's genotype. Alleles/fragment sizes of offspring and mother are tested, remaining fragments are from father. Alleged fathers identified based on inclusion. A difference in one locus can rule out father. Typical parentage test includes 8 loci. The more loci tested, the higher probability of positive identification of the father. Human identification: single locus probe systems. Analysis of one probe at a time yielded simple patterns. Able to interpret with mix of DNA from more than one individual. Used to exclude suspects. System further refined by determination gene frequency of each allele in population. The probability of two individuals having the same RFLP, or profile is less likely as more loci are analyzed. Genetic mapping: polymorphisms tend to be inherited in Mendelian pattern. Can be used as landmarks in the genome to determine location of other genes. In the presence of family history, can also demonstrate a disease has a genetic component by demonstrating close linkage to a known marker using statistical analysis. If polymorphism results in particular phenotype in individuals with disease, the more likely that the disease gene is located close to the polymorphism.

Compare and contrast different types of polymorphisms

Restriction fragment length polymorphisms (RFLPs): one or more nucleotide changes that alters DNA target of restriction enzyme Variable-number tandem repeats (VNTRs): DNA repeats of 10-50 bases Short tandem repeats: repeats of 1-10 base sequences Single nucleotide polymorphisms (SNPs): single nucleotide change in sequence.

Compare and contrast mutation detection methods based on target length, accuracy, specificity and sensitivity.

SSCP: 50-400bp, 70-100% accuracy, 80-100% specificity, 5-20% sensitivity ASO: defined target bp, 100% accuracy, 90-100% specificity, 5-20% sensitivity HR-MCA: defined target bp, 95-100% accuracy, 95-100% specificity, 1-5% sensitivity SSP: defined target bp, 98-100% accuracy, 95-100% specificity, 0.0005% sensitivity PCR-RFLP: defined target bp, 100% accuracy, 100% specificity, 0.01-1% sensitivity

Discuss sample collection, preparation, and quality control in the molecular micro lab

Sample collection: specimen handling to preserve organisms, avoid contamination, collect from original site of infection, methods are so sensitive can detect organism below level of clinical significance. Sample preparation: nucleic acid extraction procedure similar to human isolation protocols, concentration (via centrifugation), inhibitors in samples can affect amplification, sample source affects yield. Quality control: positive (confirms amp of target), sensitivity (confirms assay performs at the lower limit of detection), high/low run across range of assay, blank (contamination), non target (can amp but not target, ensures sample integrity)

Describe sequencing-based methods used for detection of point mutations and single nucleotide polymorphisms.

Sequence specific primer PCR (SSP-PCR). Primer is designed so that 3' end falls on the nucleotide to be analyzed. Template must match the primer perfectly on the 3' end in order for polymerase to create extension product. Can create different length products for mutant and wild type. High thorough pout SSP -PCR. Primers tailed at 5' end with locus specific sequences. Allele specific on one end, locus specific on the other end. PCR products amplified in a second round using CY3' or CY5' labeled primers. Hybridized to locus specific sequences on beads. Bead color (locus) and CY3 or CY5 (Allele) types the allele at each locus. Used for human haplotype project.

Describe STR structure, nomenclature, and advantages of this test method

Short tandem repeats. STR typing replaced DQA1 typing, had more discriminating power. Small STRs - efficiently amplified by PCR, including degraded DNA, only need 10 ng. Can multiplex, automate. STRs are discrete allele systems with a finite number of alleles and defined number of repeat units in tandem repeats. STR alleles are identified by PCR product size, between 100-400bp. Primers flank STR regions, size of allele PCR product representative of number of embedded repeats. Primers can be labeled with flurochromes, allows for multiplexing and detection of multiple STR loci via automated capillary electrophoresis, software detection, and analysis.

List and describe hybridization analyses to detect DNA mutations

Single strand confirmation polymorphism (SSCP): nucleic acid prefers double stranded confirmation (in the absence of complimentary strand, intra strand duplexes are formed), conformer is the 3D structure that is determined by primary sequence. Migration of conformers under very controlled denaturing and temperature conditions distinguishes sequence variants of 100-400bp. Silver stain is used because of increased sensitivity. SSCP can detect 35-200% of putative mutations (suspected mutations). Need 30% of tumor cells in sample for optimal sensitivity. Cost effective and simple technique. Allele-specific oligomer hybridization (ASO): utilizes differences melting temps of 20 bp sequences. One sequence has mismatch, the other does not. Synthetic single stranded probes w normal or mutant DNA are used, controlled stringency, detect binding. Examples include BRCA1, BRCA2, p16, cystic fibrosis screening. Replaced by microarray, bead array and NGS Melt curve analysis: post amplification analysis of real time PCR. Use dye specific for dsDNA (SYBR green, etc), initially dyes generate high signa because DNA is still double stranded, but over time, cyclic heating converts dsDNA to single stranded DNA. Sequence differences result in different melting characteristics and melting temps (Tm). Use software to convert graph to derivative of fluorescence vs temp. Normal sample should have Tm that can be distinguished from mutant. Can have issues with specificity (primer dimer, other non-specific products) High resolution melt curve analysis (MCA): uses FRET technology, specific probes hybridize next to one another. Have fluorescence when probes are next to each other (reliant on energy transfer for fluorescence). Temp increased and then probes dissociate at different temps based on wild type or mutant sequence. Can have single FRET probe variation or minor groove binder variation. Heteroduplex analysis: based on solution hybridization and electrophoretic analysis. Non identical strands of DNA are heated to complete denaturation, slowly cooled and then you have the formation of heteroduplexes. Resolve by electrophoresis. Can have heteroduplexes and homoduplexes based on sample type

Diagram a human chromosome and locate the centromere, telomere, q arm, p arm, and use basic nomenclature to characterize chromosomal aberrations

Submetacentric: divide chromosome into long and short arms. Long arm is q and short arm is p. Metacentric: create arms that are equal size Acrocentric/telocentric: centromeres very close to the ends

Interpret the results of comparative genome hybridization showing amplification or deletion

Test DNA is compared to reference DNA on metaphase spread, DNA from test and reference samples are labeled with different colored probes, Cy3 and Cy5, by nick translation. Test DNA is partially digested so that fragments will bind efficiently to denatured DNA in metaphase chromosome spread. Can identify recurrent genomic imbalances not detected by karyotyping (normally, signal should be equal. If test is higher than reference, then amplification. If test is lower than reference, then deletion). Amplified region is detected by excess test signal. Deleted region is detected by excess control signal

Compare and contrast the advantages and disadvantages of traditional (gold-standard) and molecular methods in diagnostic microbiology

Traditional: well established. Use culture to look for cytopathic effect, microscopic observation. Use serology for antibodies to virus and viral antigens. Disadvantages: Longer turn around time. Current vs past infections

Illustrate the different types of structural mutations that occur in chromosomes

Translocation: exchange of genetic material between chromosomes. Reciprocal/balanced translocations: two chromosomes break and recombine with one another, may or may not have phenotypic effects. Deletion: loss of chromosomal material, micro deletions not always detected Insertion: gain of chromosomal material Ring: deletion and joining of ends Isochromosome: transverse splitting and join of p:p or q:q parts of chromosome joined at centromere Derivative: abnormally rearranged chromosome parts joined to a normal chromosome

Define and discuss variant call file, annotation, filtering, confidence, coverage, and consensus sequence

Variant call file: text files of mutation/polymorphism that can be archived for future reference Annotation: process of identifying critical variants Filtering: single nucleotide polymorphisms are compared to previously reported variants Confidence: of variant call is based on sequencing quality and coverage Coverage: the number of times a region containing the variant is sequenced from independent fragments, also known as read depth, want at least 500X of forward, reverse sequences Consensus sequence: sequences with proportional representation of polymorphic sequences

Explain the use of immunoassays for detecting gene mutations and altered proteins

antibodies or ligands used to detect target molecules Many variations- high throughput method. Use EIA, ELISA or Immunohistochemistry Immunohistochemistry: cytology method that can be used to detect antigen in tissues, sometimes used as a screening method and usually it is lower cost

Use gene mutation nomenclature for expressing sequence changes at the DNA and protein levels

look on pgs 206-210 of coursepack

Interpret a sequencing ladder from a dideoxy chain termination (Sanger) sequencing reaction

processed in software as red (T), blue (C), black (G), or green (A) peak in electropherogam

Discuss examples of viral load testing in the microbiology and its clinical impact

qPCR and bDNA are comparable, should use same method. Determine baseline, then test at 2 to 8 weeks, and every 3 to 4 months after.

Discuss the principle of and interpret Maxam-Gilbert sequencing

requires single or double stranded DNA, one end is radioactively labeled. Labeled template is added to 4 tubes. Template is broken in each tube that uses reagents specific for characteristic breakage patterns. Following chemical reactions, fragments separated via PAGE. Have lanes with purines (A, G), pyrimidines (T, C), single bases. Read gel from bottom up. Size of fragments gives order to nucleotides. Good for short sequences. Not practical for high-thorough-put. Storage and use issues with hazardous chemicals: hydrazine and piperidine

Discuss examples of molecular diagnostic testing for respiratory and urogenital bacteria, HIV, HSV, HCV, SARS-COV2, fungi and parasites.

respiratory: various kits marketed for DNA or RNA detection, mass spec, multiplex assays urogenital: rapid, bathed, automated, hybridization and signal amplification assays HIV: serology, western blot HSV: culture, immunoassays HCV: serology, quantitative real time molecular methods SARS-COV2: PCR fungi: direct staining, isolating in culture, PCR and sequencing, mass spec parasites: direct observation of morphology, qPCR

Describe an enzymatic method for DNA sequence analysis

restriction fragment length polymorphism (RFLP). Cut amplicon containing target with appropriate restriction enzyme. Mutations may add or remove restriction site. Can be multiplexed by producing different sized targets. Evaluate for bp size band(s) to determine presence or absence of WT.

Describe the challenges and advantages of next generation sequencing (NGS) and technical items that should be considered.

sequences large numbers of templates, millions of base pairs, simultaneously at reasonable cost, reasonable turn around time. Technologies include: pyrosequencing, dye terminator sequencing, ion-conductance sequencing, single molecule sequencing, and sequencing by ligation. Library prep methods include emulsion PCR, bridge PCR, etc. Need robust computer data assembly systems to organize massive amounts of data (whole genomes and small genomes (microbial diversity)). System design - things to think about: -physical space for prep, loading, and operation of sequencers -data accumulation and storage (terabytes of large raw data sets, interface with lab info systems, electronic health records) -clinical sensitivity -data interpretation, report template design Technology types: ion conductance (pH) and reversible dye terminator sequencing. Different platforms means different technologies. Both require sequencing library prep. Library: sets of 100-500bp size fragments representing the region to be sequenced (whole genome or specific gene regions)