Genetics Quiz: Interpreting Sequencing Data

What this quiz covers

This quiz focuses on Interpreting Sequencing Data, giving you a quick way to practice the rules, question types, and explanations that matter most for Genetics.

How to use this quiz

Try each quiz question before looking at the correct answer. Use the explanations to review missed ideas, then come back to similar questions until the pattern feels familiar.

Question 1

1. A low MAPQ score indicates that the base calls within the reads are of low quality (low Phred score).
2. The variant is likely a false positive caused by reads from a repetitive element or pseudogene misaligning to this locus. (correct answer)
3. The variant is located in a region of very low sequencing coverage, making the call unreliable.
4. This is a rare and complex structural variant that standard aligners cannot place with high confidence.

Explanation: The correct answer is B. Mapping quality (MAPQ) represents the confidence that a read has been placed correctly in the reference genome. A very low MAPQ score means the aligner has found other locations in the genome where the read could align equally well (or nearly so). This is common for reads originating from repetitive regions, segmental duplications, or pseudogenes that are highly similar to functional genes. When such reads misalign, they can introduce what appear to be SNPs but are actually differences between the two genomic regions. Therefore, the variant call is highly suspect. A is incorrect; MAPQ is distinct from base quality (Phred score). A read can have perfect base calls but still have a low mapping quality. C is incorrect; the locus has 20 reads supporting the variant, so coverage itself is not necessarily low, but the quality of the alignment is poor. D is possible in some cases, but the most common and direct interpretation of uniformly low MAPQ scores for reads supporting a SNP is a misalignment issue from a homologous region.

Question 2

1. It is a true heterozygous SNP, and this pattern is a coincidence.
2. It is a true homozygous SNP, and the reference reads are misaligned.
3. It is indicative of a somatic mutation that occurred on only one chromosome.
4. It is likely a technical artifact, and the call should be rejected or filtered. (correct answer)

Explanation: When analyzing sequencing data for SNPs, you should always examine read distribution patterns across DNA strands. In high-quality sequencing, reads supporting different alleles should be randomly distributed between forward and reverse strands due to the random nature of DNA fragmentation and sequencing. The pattern described here—where all alternate allele reads map to the forward strand while all reference allele reads map to the reverse strand—represents perfect strand bias, which is extremely suspicious. This suggests a technical artifact rather than a genuine genetic variant. Such artifacts commonly arise from sequencing errors, mapping problems, or library preparation issues that systematically affect one strand differently than the other. Option A is incorrect because true heterozygous SNPs would show random strand distribution for both alleles—this level of perfect separation is virtually impossible by coincidence. Option B misinterprets the data; we see reads supporting both alleles, not evidence of homozygosity with misalignment. Option C is wrong because somatic mutations would still produce random strand distribution for the mutant allele reads, not perfect strand bias. Option D correctly identifies this as a technical artifact requiring rejection or filtering. Quality control pipelines routinely filter variants showing significant strand bias because they're unreliable. Study tip: When evaluating SNP calls, always check for strand bias as a quality metric. Real variants show roughly equal representation of alleles on both DNA strands, while technical artifacts often create systematic patterns like the one described here.

Question 3

1. The population frequency of the 'G' allele is approximately 50%.
2. The population frequency of the 'G' allele is approximately 1%.
3. The population frequency of the 'G' allele cannot be determined from this individual's data. (correct answer)
4. The 'G' allele is a rare variant because it is not the reference allele.

Explanation: The correct answer is C. The data provided is from a single individual. The ~50% ratio of 'A' and 'G' reads indicates that this individual is heterozygous. However, this says nothing about how common the 'G' allele is in the broader population. The 'G' allele could be very common (e.g., 40% population frequency) or extremely rare (e.g., <0.01% population frequency). To determine population frequency, a large cohort of individuals must be sequenced and analyzed. A is incorrect as it confuses the allele balance within a single heterozygote with the allele frequency in a population. B makes an unsupported assumption. D is incorrect because the reference allele is not necessarily the most common allele in the population; some alternate alleles are very common.

Question 4

1. The sequenced tissue exhibits somatic mosaicism for the C>G variant. (correct answer)
2. The 'G' allele is a sequencing artifact caused by PCR bias.
3. The individual is germline heterozygous for the C>G variant.
4. The sample is homozygous for the reference allele, 'C'.

Explanation: When you encounter tumor sequencing data with unexpected allele fractions, you need to consider what biological processes could produce the observed pattern. The key insight here is recognizing what a 15% variant allele fraction tells you about the cellular composition of the sample. The 15% variant allele fraction (30 G reads out of 200 total) indicates that only a subset of cells in the sample carry the C>G variant. This pattern is characteristic of somatic mosaicism, where a mutation arose during development or in adult tissue, creating a mixed population of cells - some with the original C allele and others with the acquired G variant. Answer A correctly identifies this scenario. Let's examine why the other options don't fit: Answer B suggests PCR bias, but the question states this 15% frequency is "well above the noise level," indicating it's a real biological signal, not a technical artifact. Answer C proposes germline heterozygosity, but a heterozygous individual would show approximately 50% variant allele fraction, not 15%. Answer D claims homozygosity for the reference allele, which contradicts the clear presence of G reads in the data. The fraction of variant reads directly reflects the proportion of cells carrying the mutation. In tumor samples, low variant allele fractions often indicate either normal cell contamination or subclonal mutations that only affect part of the tumor cell population. Study tip: When interpreting variant allele fractions, remember that ~50% suggests germline heterozygosity, ~100% suggests homozygosity, and intermediate percentages like 15-30% typically indicate somatic mosaicism or tumor heterogeneity.

Question 5

1. The variant is located within a gene desert and has no biological effect.
2. The variant changes an amino acid in the protein product of a gene. (correct answer)
3. The variant introduces a premature stop codon, truncating the protein.
4. The variant is located within an exon but does not change the amino acid sequence.

Explanation: The correct answer is B. Variant annotation tools predict the functional consequence of variants. A 'missense' annotation specifically means that the nucleotide change occurs within a coding region (an exon) and results in a change to the corresponding amino acid in the protein sequence (e.g., a codon for Alanine is changed to a codon for Glycine). A is incorrect; a missense variant must be in a coding region, not a gene desert. C describes a 'nonsense' or 'stop_gained' variant, which is a different, typically more severe, consequence. D describes a 'synonymous' or 'silent' variant, where the DNA change does not alter the encoded amino acid due to the redundancy of the genetic code.

Question 6

1. The DNA sample was of poor quality, leading to a high rate of random sequencing errors in that specific gene.
2. The reference genome contains a significant error or represents a rare haplotype in the region of that gene.
3. The sequencing machine malfunctioned during the part of the run corresponding to that gene.
4. The gene has a paralogous pseudogene elsewhere in the genome, causing cross-mapping of reads. (correct answer)

Explanation: The correct answer is D. Pseudogenes are non-functional copies of genes that share high sequence similarity with the functional parent gene. Reads originating from the pseudogene can incorrectly align (cross-map) to the functional gene's locus in the reference genome. Because the pseudogene has accumulated mutations over time, these differences appear as a cluster of SNPs when the reads are mapped to the functional gene. This misalignment is often flagged by low mapping quality scores, as the aligner recognizes that the reads could fit well in more than one place. A and C are unlikely because sequencing errors are typically distributed randomly across all reads and the genome, not concentrated in a single gene. B is possible, but the presence of a known pseudogene is a very common cause of such mapping artifacts and is often the first thing to check.

Question 7

1. Long-read sequencing has a much lower error rate, which is crucial for identifying SNPs.
2. Long reads can span the entire repetitive region, allowing for unambiguous mapping and variant discovery. (correct answer)
3. Short-read sequencing cannot detect single nucleotide variants, only larger structural changes.
4. Long-read sequencing generates significantly higher coverage, increasing the confidence of SNP calls.

Explanation: The correct answer is B. Short reads (e.g., 150 bp) that fall entirely within a large repetitive region (500 bp) cannot be uniquely mapped because they could have originated from multiple possible locations within the repeat. This makes variant calling in such regions difficult or impossible. Long reads, which can be thousands of base pairs long, can sequence through the entire repetitive element and include unique flanking sequences on both ends. This allows the read to be unambiguously aligned to the correct genomic location, enabling reliable identification of any variants within the repetitive sequence. A is incorrect; historically, long-read technologies have had a higher per-base error rate than short-read technologies, although this is improving. Their strength is read length, not base accuracy. C is incorrect; short-read sequencing is the most common and effective method for SNP detection in non-repetitive regions. D is incorrect; sequencing depth is a function of the amount of sequencing performed, not an inherent property of the technology type. Short-read platforms can easily generate higher coverage than long-read platforms for the same cost.

Question 8

1. The individual is likely heterozygous (T/C), but the 'T' allele was preferentially amplified during PCR.
2. The presence of the 'C' in two independent reads provides strong evidence for a true heterozygous variant.
3. The 'C' bases are likely sequencing errors, and the true genotype is homozygous reference (T/T). (correct answer)
4. The individual likely has somatic mosaicism for this variant, with the 'C' allele present in about 5% of cells.

Explanation: The correct answer is C. A Phred score of 13 is between 10 (10% error) and 20 (1% error), indicating a relatively high probability of error for those specific base calls (roughly 1 in 20, or 5%). Given that the 'C' allele is only seen in 2 out of 40 reads (5% of reads) and both instances have low quality scores, the most parsimonious explanation is that these are independent sequencing errors rather than a true biological variant. A is incorrect because while amplification bias can occur, the very low quality scores point more directly to a technical artifact. B is incorrect because it ignores the critical information provided by the low quality scores; not all reads provide equally strong evidence. D is a plausible biological explanation for a low-level allele, but sequencing error is a more likely explanation when the supporting reads have poor quality scores.

Question 9

1. At position 55249071 on chromosome 7, a homozygous 'G' allele was found with a quality score of 60.
2. At position 55249071 on chromosome 7, a heterozygous T>G variant is present, supported by approximately 30 reads. (correct answer)
3. A gene on chromosome 7 has a pathogenic T>G mutation with an allele frequency of 50% in the population.
4. The reference allele is 'T' and the alternate allele is 'G'; this variant is likely an error as the quality score (225) is too low.

Explanation: The correct answer is B. The VCF line indicates the chromosome (chr7), position (55249071), reference allele (T), and alternate allele (G). The GT (Genotype) field 0/1 indicates a heterozygous state (one reference allele '0' and one alternate allele '1'). The DP=60 indicates a total read depth of 60, and AF=0.5 indicates an allele frequency of 0.5 within the sample's reads. Therefore, the alternate allele 'G' is supported by approximately 0.5 * 60 = 30 reads. A is incorrect because 0/1 signifies a heterozygous, not homozygous, genotype (1/1). The quality score is 225, not 60. C is incorrect because AF=0.5 refers to the allele frequency within this single sample's reads, not the population frequency, and VCF files do not inherently contain information about pathogenicity. D is incorrect because a QUAL score of 225 is extremely high (Phred-scaled probability of error is 10^(-22.5)), indicating very high confidence in the variant, not an error.

Question 10

1. The population frequency of the 'G' allele is approximately 50%.
2. The population frequency of the 'G' allele is approximately 1%.
3. The population frequency of the 'G' allele cannot be determined from this individual's data. (correct answer)
4. The 'G' allele is a rare variant because it is not the reference allele.

Explanation: The correct answer is C. The data provided is from a single individual. The ~50% ratio of 'A' and 'G' reads indicates that this individual is heterozygous. However, this says nothing about how common the 'G' allele is in the broader population. The 'G' allele could be very common (e.g., 40% population frequency) or extremely rare (e.g., <0.01% population frequency). To determine population frequency, a large cohort of individuals must be sequenced and analyzed. A is incorrect as it confuses the allele balance within a single heterozygote with the allele frequency in a population. B makes an unsupported assumption. D is incorrect because the reference allele is not necessarily the most common allele in the population; some alternate alleles are very common.

Question 11

1. The high number of identical reads indicates a tandem duplication event at this locus.
2. The identical reads are likely PCR duplicates, which artificially inflate the evidence for the variant. (correct answer)
3. This pattern confirms the variant is real, as it has been captured by multiple independent sequencing events.
4. The reads with identical coordinates must be from a different, but highly similar, genomic region.

Explanation: The correct answer is B. During library preparation for NGS, PCR is used to amplify DNA fragments. If the same original DNA fragment is amplified multiple times, it will result in a set of reads that have the exact same start and end points. These are known as PCR duplicates. Standard bioinformatics pipelines identify and often remove or down-weight these reads because they do not represent independent evidence from the original sample. Their presence can artificially inflate the read count supporting a particular allele, potentially leading to a false positive variant call. A is incorrect because while tandem duplications can be complex to sequence, this specific read pattern is the classic signature of PCR duplicates. C is incorrect because the identical start/end points suggest the reads are not from independent events. D is less likely; while misalignment can happen, the presence of other reads with variable coordinates that map correctly makes PCR duplication the more direct explanation for the identical subset.

Question 12

1. Both variants are called with equal confidence because their alternate allele frequencies are both approximately 50%.
2. Variant 1 is called with higher confidence because the 50/50 allele ratio is more exact than for Variant 2.
3. Variant 2 is called with higher confidence due to its significantly greater sequencing depth. (correct answer)
4. Neither variant can be called confidently, as true heterozygous variants require thousands of reads for support.

Explanation: The correct answer is C. Sequencing depth, or coverage, is a critical factor in the confidence of a variant call. While both variants show an allele ratio consistent with a heterozygous state (~50%), Variant 2 is supported by a much larger number of independent reads (100 total reads vs. 8). The higher depth for Variant 2 makes it much less likely that the observed pattern is due to random sequencing errors or PCR amplification bias, thus yielding a higher confidence call. A is incorrect because it ignores the crucial role of sequencing depth. B is incorrect because the small deviation from a perfect 50/50 ratio in Variant 2 is expected due to random sampling effects during sequencing, and it does not reduce confidence compared to the low-depth Variant 1. D is an overstatement; while more depth is generally better, confident calls can often be made with depths well below thousands of reads, and 100x is generally considered very good coverage.

Question 13

1. The variant is a false positive resulting from reads from GENEB misaligning to the GENEA locus. (correct answer)
2. The individual has a gene conversion event where part of GENEB replaced part of GENEA.
3. The variant represents a de novo mutation that occurred in GENEA.
4. The variant is real, and the low mapping quality is due to the gene's location in a GC-rich region.

Explanation: When you encounter questions about variant calling in genomic regions with paralogs, you need to consider how sequence similarity can create technical artifacts in sequencing data analysis. The key clue here is that reads supporting the variant have low mapping quality yet match GENEB perfectly. This pattern strongly indicates misalignment. During sequence alignment, reads originating from GENEB are being incorrectly mapped to the GENEA locus because of high sequence similarity between the paralogs. When the aligner encounters reads that could map to multiple locations with similar scores, it assigns low mapping quality scores to reflect this uncertainty. The "variant" called in GENEA is actually just the normal sequence difference between GENEA and GENEB, creating a false positive variant call. Option A correctly identifies this misalignment artifact. Option B (gene conversion) would result in reads that actually originated from the GENEA locus, not reads that perfectly match GENEB, and wouldn't necessarily show the low mapping quality pattern described. Option C (de novo mutation) fails to explain why the reads perfectly match GENEB rather than showing a novel sequence change. Option D incorrectly attributes low mapping quality to GC content rather than the more obvious explanation of paralog interference. For genetics exam success, remember that low mapping quality combined with perfect matches to known paralogs is a classic red flag for misalignment artifacts. Always consider technical causes before biological explanations when the sequencing quality metrics don't support the variant call.

Question 14

1. Sanger data would show two distinct bands at the same position on a gel, while NGS would show a 50/50 ratio of base calls.
2. Sanger data would show a single chromatogram peak of intermediate height, while NGS would show two distinct clusters on a plot.
3. Sanger data would show two overlapping fluorescent peaks at the same position in a chromatogram, while NGS would show a mix of reads supporting two different bases. (correct answer)
4. Sanger data would produce a single clean sequence read of the dominant allele, while NGS would detect the minor allele from the other chromosome.

Explanation: The correct answer is C. Sanger sequencing produces an electropherogram (chromatogram) where each base is represented by a fluorescent peak. At a heterozygous position, the sequencer detects both nucleotides simultaneously, resulting in two overlapping peaks of different colors, each at roughly half the height of a homozygous peak. In contrast, NGS sequences individual DNA molecules. For a heterozygous SNP, this results in a collection (stack) of short reads, where approximately half of the reads contain one allele and the other half contain the second allele. A is an outdated description of slab gel sequencing; modern Sanger uses capillary electrophoresis and chromatograms. B is incorrect; Sanger shows two overlapping peaks, not one of intermediate height. D is incorrect; Sanger sequencing is fully capable of detecting heterozygous variants; it does not just sequence the 'dominant' allele.

Question 15

1. A high rate of sequencing error at the ends of the reads.
2. The presence of a structural variant breakpoint, such as an insertion or translocation. (correct answer)
3. These reads are too short to be mapped confidently by the alignment software.
4. Contamination of the sample with DNA from another species.

Explanation: The correct answer is B. Soft-clipping is a strategy used by aligners when a portion of a read matches a reference location, but the remainder of the read does not. When multiple reads show a consistent soft-clipped pattern at the same location, it provides strong evidence for a structural variant. The mapped portion of the read anchors it to a genomic location, and the soft-clipped portion represents the sequence from the other side of a breakpoint. For example, it could be the sequence of a large insertion that is not present in the reference, or sequence from a different chromosome in the case of a translocation. A is incorrect because sequencing errors are typically random, whereas this pattern describes a consistent sequence in the clipped ends. C is incorrect because a portion of the reads did map confidently. D is possible, but a structural variant is a more common and direct interpretation of this specific alignment signature.

Question 16

1. 0/0
2. 0/1
3. ./.
4. 1/1 (correct answer)

Explanation: When you encounter VCF (Variant Call Format) files in genetics, understanding the genotype (GT) field notation is crucial for interpreting genomic data. The GT field uses a numerical system where each number represents a specific allele: 0 refers to the reference allele, and 1 refers to the alternate allele. For a homozygous alternate genotype, the individual has two copies of the alternate allele (T in this case, rather than the reference C). Since both alleles are alternate alleles, this is represented as 1/1 in the GT field - each "1" indicates one copy of the alternate allele T. Looking at the incorrect options: A) 0/0 represents a homozygous reference genotype, meaning the individual has two copies of the reference allele C. B) 0/1 indicates a heterozygous genotype, where the individual has one reference allele (C) and one alternate allele (T). C) ./. represents missing or unknown genotype data - the periods indicate that the genotype could not be determined from the sequencing data. The correct answer is D) 1/1, which definitively indicates two copies of the alternate allele T. Study tip: Remember the VCF numbering system: 0 = reference allele, 1 = first alternate allele. Count the numbers to determine zygosity - same numbers mean homozygous, different numbers mean heterozygous, and periods mean missing data. This notation appears frequently in genomics coursework and research.

Question 17

1. The variant is a germline de novo mutation that arose in the sperm or egg cell that formed the child. (correct answer)
2. The stated paternity is incorrect, and the child inherited the variant from the true biological father.
3. The variant is a false positive in the child due to a sequencing artifact.
4. Both parents are mosaic for the mutation at a level below the limit of detection for sequencing.

Explanation: When you encounter trio sequencing data showing a variant in the child but not the parents, you're dealing with a classic scenario that tests your understanding of de novo mutations and their detection limits. The most straightforward interpretation is that this represents a germline de novo mutation (A). With high coverage (>30x) and excellent quality scores, the sequencing data is reliable. The variant likely arose spontaneously in either the sperm or egg cell that contributed to this child's formation. De novo mutations occur at a rate of approximately 1-2 × 10⁻⁸ per nucleotide per generation in humans, making this a well-documented biological phenomenon. Let's examine why the other options are less supported: Option B (incorrect paternity) would require the true father to carry this specific variant, but you'd need additional evidence beyond this single locus to support such a conclusion. Option C (sequencing artifact) is unlikely given the explicitly stated high coverage and excellent quality scores - these parameters are specifically mentioned to rule out technical issues. Option D (parental mosaicism below detection limits) is theoretically possible but represents a more complex explanation. While low-level mosaicism can escape detection, it's less probable than a straightforward de novo event. The key principle here is parsimony: when sequencing quality is high and coverage is adequate, the simplest explanation for a child-only variant is a de novo mutation. Remember that de novo mutations are a normal part of human genetics - each person carries roughly 50-100 new mutations not present in their parents.

Question 18

1. Both variants are called with equal confidence because their alternate allele frequencies are both approximately 50%.
2. Variant 1 is called with higher confidence because the 50/50 allele ratio is more exact than for Variant 2.
3. Variant 2 is called with higher confidence due to its significantly greater sequencing depth. (correct answer)
4. Neither variant can be called confidently, as true heterozygous variants require thousands of reads for support.

Explanation: The correct answer is C. Sequencing depth, or coverage, is a critical factor in the confidence of a variant call. While both variants show an allele ratio consistent with a heterozygous state (~50%), Variant 2 is supported by a much larger number of independent reads (100 total reads vs. 8). The higher depth for Variant 2 makes it much less likely that the observed pattern is due to random sequencing errors or PCR amplification bias, thus yielding a higher confidence call. A is incorrect because it ignores the crucial role of sequencing depth. B is incorrect because the small deviation from a perfect 50/50 ratio in Variant 2 is expected due to random sampling effects during sequencing, and it does not reduce confidence compared to the low-depth Variant 1. D is an overstatement; while more depth is generally better, confident calls can often be made with depths well below thousands of reads, and 100x is generally considered very good coverage.

Question 19

1. The high number of identical reads indicates a tandem duplication event at this locus.
2. The identical reads are likely PCR duplicates, which artificially inflate the evidence for the variant. (correct answer)
3. This pattern confirms the variant is real, as it has been captured by multiple independent sequencing events.
4. The reads with identical coordinates must be from a different, but highly similar, genomic region.

Explanation: The correct answer is B. During library preparation for NGS, PCR is used to amplify DNA fragments. If the same original DNA fragment is amplified multiple times, it will result in a set of reads that have the exact same start and end points. These are known as PCR duplicates. Standard bioinformatics pipelines identify and often remove or down-weight these reads because they do not represent independent evidence from the original sample. Their presence can artificially inflate the read count supporting a particular allele, potentially leading to a false positive variant call. A is incorrect because while tandem duplications can be complex to sequence, this specific read pattern is the classic signature of PCR duplicates. C is incorrect because the identical start/end points suggest the reads are not from independent events. D is less likely; while misalignment can happen, the presence of other reads with variable coordinates that map correctly makes PCR duplication the more direct explanation for the identical subset.

Question 20

1. A low MAPQ score indicates that the base calls within the reads are of low quality (low Phred score).
2. The variant is likely a false positive caused by reads from a repetitive element or pseudogene misaligning to this locus. (correct answer)
3. The variant is located in a region of very low sequencing coverage, making the call unreliable.
4. This is a rare and complex structural variant that standard aligners cannot place with high confidence.

Explanation: The correct answer is B. Mapping quality (MAPQ) represents the confidence that a read has been placed correctly in the reference genome. A very low MAPQ score means the aligner has found other locations in the genome where the read could align equally well (or nearly so). This is common for reads originating from repetitive regions, segmental duplications, or pseudogenes that are highly similar to functional genes. When such reads misalign, they can introduce what appear to be SNPs but are actually differences between the two genomic regions. Therefore, the variant call is highly suspect. A is incorrect; MAPQ is distinct from base quality (Phred score). A read can have perfect base calls but still have a low mapping quality. C is incorrect; the locus has 20 reads supporting the variant, so coverage itself is not necessarily low, but the quality of the alignment is poor. D is possible in some cases, but the most common and direct interpretation of uniformly low MAPQ scores for reads supporting a SNP is a misalignment issue from a homologous region.