Next Generation Sequencing Midterm

E

A correct order of transcriptome analysis would be: A) Sequencing, Alignment, Variant call B) Sequencing, Alignment, Expression-abundance assessment C) Variant call, Alignment, Expression abundance assessment D) Expression abundance assessment, Alignment, Variant call E) A and B

C

Bisulfite sequencing is a well-established protocol to detect ______________________ in genomic DNA A) acetylated cytosines B) acetylated guanines C) methylated cytosines D) methylated guanines

D

Bridge PCR in Illumina machines: A) Results in hundreds of millions of unique clusters B) Occurs on the surface of an Illumina flow cell C) Involves coating the surface of the flow cell with a lawn of two distinct oligonucleotides D)All of the above

D

Chip-Seq is based on: A) Degradation of naked DNA sequences B) DNA-protein interactions C) Protection of DNA from degradation D) All of the above E) None of the above

B

Dideoxy sequencing is also known as chain termination sequencing because A) The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a free 5' carbon B) The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a free 3' OH C) The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a nitrogencontaining base (e.g. A,T, C, or G) D) Chain termination is the same as sequencing by synthesis

A

Exome sequencing comes in two major varieties: hybridization-based capture and multiplex PCR. Which has higher throughput? A) Hybridization-based capture B) Multiplex PCR C) Neither have high throughput D) Both have high throughput

B

Exome sequencing comes in two major varieties: hybridization-based capture and multiplex PCR. Which would be used to identify difficult targets? A) Hybridization-based capture B) Multiplex PCR C) Neither would be suitable D) Both would be suitable

D

High expression of mitochondrial genes in a scRNA-Seq experiment could be an indicator of: A) Poor sample quality B) High fraction of apoptotic or lysing cells C) Biology of the particular sample ie. tumor D) All of the above

B

Homopolymers are a major challenge for: A) The Illumina HiSeq machine B) The Roche 454 machine

B

How do second generation sequencing technologies make up for their low accuracy per read compared to Sanger sequencing? A) 2nd generation technologies use very long reads B) 2nd generation technologies use multiple reads C) There is no need to make up for "low accuracy", 2nd generation technologies are far more accurate than Sanger sequencing D) None of the above

E

Human exome is comprised of: A) ~180,000 exons B) ~ 30 megabases DNA C) ~1% of the human genome D) Contains >80% of the disease causing mutations E) All of the above

A

Illumina and 454 sequencing are based on A) sequencing by synthesis B) chain termination C) nanopores

D

In Hi-C, quantitation of chromatin interactions is achieved through A) Sanger sequencing B) ChIP-seq C) Computational methods D) Massively parallel deep sequencing

B

In the sequencing-through-synthesis setting, how could you ensure DNA-chain elongation by only one nucleotide at a time? A) Add one type of nucleotide per each elongation cycle B) Use reversible nucleotide terminators C) Control the time of the elongation cycle D) Label the four nucleotides differently E) Use quantitative detection of the signal

B

Large variations like chromosome copy number A) Will be detected by exome sequencing B) Might not be detected by exome sequencing

B

Oligonucleotides are: A) Chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing B) Short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology and synthetic biology applications C) Genes that used in reconstructing phylogenies, due to their slow rates of evolution D) Messengers carrying instructions from DNA for controlling the synthesis of proteins

D

Pacific Biosciences SMRT technology: A) Relies on reversible terminators B) Uses only two different nucleotide labels C) Creates clusters of DNA molecules, separated into wells D) Uses fluorescent labels on the terminal phosphate and captures the flash of light

D

The 454 Roche machine: A) Uses reversible terminators B) Uses labelled dNTPs C) Measures the color of the signal to determine base pair addition D) Adds dNTPs sequentially (only one is present in any given moment)

C

The machine marked C is the: A) Hiseq Illumina B) 454 Roche C) SOLiD ABI 2007 D) None of the above

D

The plot shown is a histogram illustrating the number of genes expressed per cell in a scRNA-Seq experiment. Knowing this fact, which group (1, 2, or 3) is probably composed of cells with are actively proliferating A) Group 1 B) Group 2 C) Group 3 D) Cannot be determined from the information provided

B

The schematic shown matches the procedures that are part of which sequencing technology? A) Hi-C Seq B) ChIP-seq C) RNA-seq D) 3rd generation sequencing

D

This is a targeted RNA sequencing method that is able to provide higher sequencing coverage for selected regions of the genome. It is highly suitable for discovering novel exons, genes, and splice isoforms but requires a large amount of total RNA for capture. A) DNase-Seq B) Bisulfite sequencing C) ATAC-Seq D) CaptureSeq

A

This sequencing technology detects chromatin interaction both within and between chromosomes by covalently crosslinking protein/DNA complexes with formaldehyde A) Hi-C Seq B) ChIP-seq C) RNA-seq D) 3rd generation sequencing

D

This technique can detect RNA-modifications. In it, m6A-specific antibodies are used to immunoprecipitate RNA which is then reverse transcribed and sequenced. A) DNase-Seq B) Bisulfite sequencing C) HITS-CLIP/CLIP-Seq/PTB-Seq D) MeRIP-Seq

C

This technique can identify RNA-protein interactions. RNA-protein complexes are UV-crosslinked and immunoprecipitated. RNA is then extracted, reverse transcribed, and sequenced. A) DNase-Seq B) Bisulfite sequencing C) HITS-CLIP/CLIP-Seq/PTB-Seq D) CaptureSeq

B

This technique can provide structural information about RNA. A unique barcode is added to RNA and then the molecule is allowed to fold under pre-established in vitro conditions. Then the RNA is treated with a reagent, reverse transcribed, and sequenced. The length distribution of reads reflects the location of the modifications A) DNase-Seq B) SHAPE-Seq C) HITS-CLIP/CLIP-Seq/PTB-Seq D) MeRIP-Seq

B

This type of sequencing involves the single addition of a dNTP in limited amounts. Upon incorporation, DNA polymerase pauses. The process of incorporation releases a pyrophosphate, which gets converted to ATP, which then participates in converting luciferin to oxyluciferin. The whole process emits light proportionately to the amount of ATP involved (and therefore the number of nucleotides which were incorporated). The order and intensity of the light peaks are recorded, as illustrated by the attached figure. A) true Single Molecule Sequencing (tSMS) B) Pyrosequencing C) single-molecule real-time sequencing (SMRT) D) Exome sequencing

True

True or False: Full length scRNA-Seq methods like SmartSeq2 have no UMIs so amplification bias is harder to control

False

True or False: Most microorganisms can be cultivated on a nutrient medium

True

True or false: Small RNA sequencing and analysis is an emerging new field; small RNAs usually require separate library prep and analysis, as compared to the full size RNAs

C

When doing a cluster analysis in scRNA-Seq, doublets are: A) Represented in distinct clusters B) Represented by a streak between clusters C) Either A or B D) None of the above

A

Whereas the 2nd generation Roche 454 measures pyrophosphate, the 3rd generation Ion Torrent by Life Technologies measures A) pH (via H+ molecules) as bases are incorporated B) light (via luciferase) as bases are incorporated C) color (via tagged nucleotides) as bases are incorporated D) radioactivity (via radiolabeled nucleotides) as bases are incorporated

D

Which gene is often highly expressed and correlated with high mitochondrial coexpression. It is used as a proxy for mitochondrial expression in scRNA-seq A) COI B) 16S C) RNAG 1 D) MALAT 1

B

Which of the following "omes" relates to the DNA sequence of expressed genes? A) Genome B) Exome C) Proteome D) Metabolome

D

Which of the following are commonly used in 2nd generation sequencing technologies to ensure the addition of only one nucleotide per cycle: A) Barcoding B) Bridge PCR C) Oligonucleotide adaptors D) Reversible terminators

D

Which of the following can create a doublet signature in scRNA-Seq analysis?: A) Refrozen samples B) Low quality of cells C) Two cells in a single droplet D) All of the above

D

Whole exome sequencing: A) Costs 1/3 as much as whole genome sequencing B) Generates 1/15 the amount of data as whole genome sequencing C) Is easier to analyze, computationally D) All of the above

E

FASTQ is A) The format of the raw sequence generated through next gen sequencing platforms B) Sequence of nucleotides with quality data for each of them C) A text-based format for storing a biological sequence and its quality information D) The file that we use to align to a reference sequence E) All of the above

A

Fill in the blank: In humans as a diploid organism, each individual carries two alleles for each genomic variant. Both alleles (homozygous or heterozygous), contribute to the individual's phenotype. However, an imbalanced expression of the two alleles negates the equal contribution of the alleles to the phenotype. _______________________ is considered when the expressed genes exhibit unbalanced expression from the two alleles with one allele expresses in access relative to the other A) Allelic Specific Expression (ASE) B) Gene dosage C) Copy number variation D) RNA editing

D

Fill in the blank: Libraries for scRNA-seq are typically generated via cell lysis, reverse transcription into first-strand cDNA using uniquely _____________ beads, second-strand synthesis, and cDNA amplification A) Hybridized B) Crosslinked C) Immunoprecipitated D) Barcoded

D

Fill in the blank: There are _________ times more cells you can observe microscopically than colonies you can grow on a petri dish. A) 2 B) 5 C) 10 D) 100

D

Filtering of genes prior to clustering frequently includes: A) Genes with little or no variation across the dataset B) Mitochondrial genes C) genes whose expression accounts for a large proportion of the expression (thus skewing the distribution of the remaining genes) D) All of the above

C

In massively parallel sequencing, adaptors serve all of the following purposes except: A) They are used as primers for PCR B) They attach to a target (hybridization) C) They are used to cleave DNA into smaller fragments D) They can be used as barcodes

B

In regards to scRNA-Seq methods, spike-ins are often used in: A) Droplet-based platforms B) Non-droplet-based platforms

B

In scRNA-Seq, a high content of mitochondrial RNA may indicate: A) Doublets B) Apoptosis C) Spike-ins D) 3' bias

B

NGS technologies are best defined by A) The fact that they sequence by synthesis B) The fact that they are high-throughput C) The fact that they require amplification D) None of the above

E

Pacific Biosciences SMRT technology: A) Is useful for creating de novo assemblies B) Can identify alternatively spliced isoforms in transcriptome sequencing C) Is often used in conjunction with short-read technology in hybrid sequencing approaches D) Can detect base modifications, such as methylation E) All of the above

D

QC for scRNA-seq often includes: A) Filtering of low quality cells B) Filtering of low-quality genes C) Filtering out effects of doublets and Ambient RNAs D) All of the above

D

The Illumina HiSeq machine: A) Sequentially adds dNTPs B) Relies on binary signal detection (either a yes or no based on base pair addition) C) Relies on order of base pair addition, not color D) Uses reversible terminators

C

The gray dots in this plot are probably: A) Ambient RNA molecules B) Chimera cells C) Doublets D) Transcriptional bursting

B

The image shown best corresponds to which of the following quality control considerations for scRNA-Seq: A) Drop-outs B) Transcriptional bursting C) Amplification bias D) Dilution

B

The left panel of the image most accurately describes: A) Next generation sequencing B) Sanger sequencing C) Hi-C D) Microbiome sequencing E) All of the above (Lecture 1)

B

The machine marked A is the: A) Hiseq Illumina B) 454 Roche C) SOLiD ABI 2007 D) None of the above

A

The machine marked B is the: A) Hiseq Illumina B) 454 Roche C) SOLiD ABI 2007 D) None of the above

C

The plot shown is a histogram illustrating the number of genes expressed per cell in a scRNA-Seq experiment. Knowing this fact, which group (1, 2, or 3) is probably composed of multiple cells (ie doublets) A) Group 1 B) Group 2 C) Group 3 D) Cannot be determined from the information provided

B

The plot shown is a histogram illustrating the number of genes expressed per cell in a scRNA-Seq experiment. Knowing this fact, which group (1, 2, or 3) is probably composed of the cells which we want to analyze (single cells with a successful run) A) Group 1 B) Group 2 C) Group 3 D) Cannot be determined from the information provided

A

The plot shown is a histogram illustrating the number of genes expressed per cell in a scRNA-Seq experiment. Knowing this fact, which group (1, 2, or 3) is probably composed of those which had a failed library A) Group 1 B) Group 2 C) Group 3 D) Cannot be determined from the information provided

A

This technique provides accurate representation of the location of regulatory proteins in the genome. It can detect "open" chromatin, no prior knowledge of the sequence or binding protein is required, and it has great sensitivity at promoters. A) DNase-Seq B) Bisulfite sequencing C) ATAC-Seq D) CaptureSeq

C

This technique uses the Tn5 transposome to detect nucleosome-free regions of the genome. A) DNase-Seq B) Bisulfite sequencing C) ATAC-Seq D) CaptureSeq

False (Any amplification step in a scRNA-Seq experiment will introduce bias in the data but methods which use UMIs will control this to a large extent)

True or False: Any amplification step in a scRNA-Seq experiment will introduce bias in the data, especially in methods which use Unique Molecular Identifiers (UMIs)

False (High quality scRNA-Seq data will show that a majority of the cells have <7% mt content)

True or False: High quality scRNA-Seq data will show that a majority of the cells have >7% mt content

True

True or False: If the QC-measures are different between batches, it is likely that batch effects are strongly affecting your data and you need to remove them

False (In scRNA-Seq, empty droplets can be used to estimate a background signal, giving researchers the ability to remove ambient RNA contamination)

True or False: In scRNA-Seq, empty droplets can be used to estimate a background signal, giving researchers the ability to remove mitochondrial RNA contamination

True

True or false: Cell debris may cause doublet signatures

True

True or false: Each cluster on an Illumina flow cell produces a single sequencing read. Therefore, 10,000 clusters on the flow cell would produce 10,000 single reads and 20,000 paired-end reads.

False (PCR may introduce sequencing errors. A high number of cycles increases the probability of amplifying these errors)

True or false: High-fidelity Taq polymerase eliminates any chance of introducing sequencing errors in PCR

False (Optimal lysis conditions may vary from celltype to celltype and for nuclei vs cells)

True or false: In scRNA-Seq, lysis procedures are standard across all cell types

True

True or false: In the Illumina platform the signal produced by the synthesis of one dNTP on a strands is not enough to be detected. This is why it is necessary to amplify the DNA sequences and produce a dense amount of sequences per area on the flow cell.

True

True or false: Prior to sequencing, 1st and 2nd generation technologies require amplification, often by PCR

C

Which of the following best describes the key difficulty associated with the cell dissociation step in scRNA-Seq? A) Laser capture microscopy often causes cells to initiate transcriptional bursting B) Nuclei sorting is a difficult process because of the differing size of nuclei C) Incomplete dissociation leaves cells stuck together but harsh dissociation damages cells, degrades RNA, and causes RNA to leak which introduces noise D) All of the above

E

Which of the following can be identified by whole exome sequencing?: A) Non-genic control elements B) mitochondrial DNA C) Structural DNA elements D) Copy number variation E) None of the above can be identified

E

Which of the following disadvantages are associated with Helicos Biosciences true Single Molecule Sequencing (tSMS) A) The time to sequence a single nucleotide is high B) Read lengths are 25-32 nucleotides long C) High noise and high error rate D) Heavy use of reagents (ie. high price) E) All of the above

D

Which of the following disadvantages are associated with Pacific Biosciences SMRT technology A) The time to sequence a single nucleotide is high B) Read lengths are only 25-32 nucleotides long C) Especially hard to sequence repetitive regions D) Cost and throughput

D

Which of the following does an ion torrent machine use? A) Scanning B) Cameras C) Dyes D) None of the above

E

Which of the following interactions between protein and DNA would not be identified through the use of ChIP-seq A) Transcription factors B) Histones C) RNA polymerase D) DNA polymerase E) RNA editing F) DNA repair enzymes

B

Which of the following is a non-electrophoretic, bio-luminescence method that measures the release of inorganic pyrophosphate by proportionally converting it into visible light using a series of enzymatic reactions A) true Single Molecule Sequencing (tSMS) B) Pyrosequencing C) single-molecule real-time sequencing (SMRT) D) Exome sequencing

E

Which of the following is a problem with exome sequencing? A) Not all genes are recognized B) Not all targeted exons are well captured C) Not all targeted sequences can be aligned D) Not all aligned sequences can be correctly called E) All of the above are problems

B

Which of the following is characterized by being the "Dominant platform, both in terms of installed base and bioinformatics development efforts"? A) 454 Roche B) HiSeq Illumina C) SOLiD ABI D) This applies equally to all of the above

C

Which of the following is false regarding the sequencing step of an Illumina-based experiment? A) There are three steps - incorporation, fluorescence imaging, and photochemical cleavage B) Only one nucleotide is added per sequencing cycle C) The signal is quantitative, not based on color D) All of the above are true

C

Which of the following is not a benefit associated with 3rd generation single molecule sequencing technologies? A) Greatly reduced input material B) Simplified library prep C) Increased Data volume D) Increased read length E) Optimized bioinformatics F) Fidelity G) Reduced run time

B

Which of the following is not a major type of PCR used in 2nd generation sequencing technologies? A) Bridge PCR B) Pyro PCR C) Emulsion PCR D) All of the above are used

G

Which of the following is not a typical step in 2nd generation sequencing technologies? A) Fragmentation B) Adaptor ligation C) Fragment segregation D) PCR- amplification E) Sequencing F) Signal detection G) All of the above are typical steps

C

Which of the following is not an example of third generation, single molecule sequencing: A) Single Molecule Real Time Sequencing (PacBio) B) True Single Molecule Sequencing (Helicos) C) Pyrosequencing (454 Roche) D) Oxford nanopore

C

Which of the following is not one of the artifacts associated with the dissociation step of scRNA-Seq? A) Biased cell populations because some cell populations may be more difficult to dissociate B) Dissociation protocols may introduce transcriptional changes C) Dissociation can cause amplification biases due to activation of reverse transcriptase D) All of the above are artifacts associated with the dissociation step of scRNA-Seq

D

Which of the following is not typically a reason to do transcriptome sequencing?: A) To determine gene-expression patterns characteristic for tissue, organism, condition, etc B) To identify genes/isoforms expressed differentially between normal and disease state C) To identify novel spliced isoforms D) To find "THE GENE" responsible for a disease/phenotype E) To study splicing

E

Which of the following must be considered in the normalization step of a scRNA-seq experiment? A) Capture and RT efficiency B) Dropout / amplification bias C) Dilution factor D) Sequencing amount E) All of the above

D

Which of the following sequencing methods is not correctly matched with the company that develops it: A) true Single Molecule Sequencing (tSMS) - Helicos Biosciences B) single-molecule real-time sequencing (SMRT) - Pacific Biosciences C) Ion Torrent - Life Technologies D) Nanopore sequencing - Illumina

A

Which of the following sequencing methods relied on bacterial cloning at one point? A) Sanger sequencing B) Massively parallel sequencing C) Hi-C-Seq D) CHiP-Seq

D

Which of the following sequencing technologies require amplification?: A) Sanger sequencing B) Massively parallel cyclic sequencing C) Single molecule sequencing D) Both A and B (Lecture 1)

C

Which of the sequencing technologies combines high-throughput sequencing with chromatin immunoprecipitation to identify specific protein-DNA interactions genome-wide: A) Exome sequencing B) Hi-C sequencing C) ChIP-seq D) None of the above

A

Which of the third generation sequencing technologies uses a glass surface covered with a multi "T" single-stranded DNA sequence, captures DNA strands that are prepared with a multi "A" end, does not use colonies, and uses the alternate addition of fluoro-tagged nucleotides one at a time to capture sequences? A) Helicos Biosciences B) Pacific Biosciences C) Ion Torrent/Ion proton D) Nanopore

D

Which of the third generation sequencing technologies uses the conductance of nucleic acids to provide a readout? A) Helicos Biosciences B) Pacific Biosciences C) Ion Torrent/Ion proton D) Nanopore

B

Which plot depicts better scRNA-Seq data based on the mt content? A) Left group B) Right group C) Cannot be determined D) Both A and B are equally good

D

Which term is best defined as: A pool of mRNA molecules that have been released in the cell suspension, likely from cells that are stressed or have undergone apoptosis. Cross-contamination occurs when this pool of molecules gets incorporated into droplets and is barcoded and amplified along with a cell's native mRNA. Contamination from this source is evident when highly-expressed cell-type specific genes are observed at low levels in other cell populations. A) Mitochondrial RNA B) Laser capture RNA C) Degraded RNA D) Ambient RNA

B

Which term is best defined as: External molecules added in a known concentration as a control to model technical noise, drop-out rates, and starting amount of RNA in the cell. They are useful for data normalization but can behave differently than endogenous genes and cannot be used in drop-seq methods. A) Doublets B) Spike-in RNAs C) Ambient RNA D) Cell debris

D

Which three sequencing projects are NOT based on targeted sequencing? A) Microbiome, Exome, ChIP-Seq B) Chip-Seq, Exome, Hi-Seq C) Sanger, Transcriptome, ChIP-Seq D) Genome, Transcriptome, Microbiome E) Transcriptome, Hi-Seq, ChIP-seq

C

Which two technologies employ similar principles based on the natural release of molecules during the addition of every next nucleotide? A) Sanger and Solid B) PacBio and Nanopore C) 454 and Ion Torrent/Proton D) Helicos and Illumina E) There are no such two technologies listed

C

Which variations/variation features can not be captured by transcriptome sequencing? A) RNA editing B) Allele-specific expression C) DNA-protein binding D) Monoallelic expression (imprinting, transcription factors, methylation) E) Loss of heterozygosity F) Somatic over expressed (SOM-E) or lost (SOM-L)

E

You are looking for mutations that generate new binding motifs for the RNA-binding protein X, and you do not know all the sequence motifs recognizable by X. Your approach includes: A) Precipitation with X-specific antibody and RNA-seq B) RNA-seq C) Variant call D) Identification of variants different from the reference genome sequence E) All of the above

A

scRNA-seq platforms typically support either: A) Full-length transcript sequencing or Unique Molecular Identifiers (UMI) B) Micromanipulation or FACS C) Microfluidic technology or CellSearch D) Unique Molecular Identifiers (UMI) or micromanipulation