Monivalinta 3

Library preparation means in sequencing technology the operation of creating all possible sequences of 35 bases (if reads of 35 bp will be sequenced) which can be compared to the actual reads coming from the expereiment. True or false?

false

Nanopore sequencing is based on observing tiny pores across which there is a voltage, so that an electric current runs through them as ions migrate in the solution. The previous statemetns are correct, and you only need to evaluate what follows. DNA you want to sequence is being copied by DNA polymerase, and the newly formed copy strand passes through the pore. Depending on which bases are going through the pore, the currecnt is obstructed by varying degrees so that the pattern of changes in the current can be interpreted into the sequence. True or false?

tru

Barcoding means in sequencing technology the addition of a specific sequence into each fragment you intend to sequence. In this way, multiple samples can be mized and sequenced together, because the reads belonging to each sample can be sorted after the sequencing run is finished, based on finding the barcode sequence at the start of the fragment. True or false?

true

Doctors in your team think that a patient may have large-scale structural variants as a cause for the symptoms they display. What would be the most appropriate sequencing approach to find out if this is true?

a. Full-genome sequencing with a short-read method because it is cheapest

b. Exome sequencing

c. Full-genome sequencing with a short-read method because it is the only method available at the local facility

d. RNA-seq with a long-read method

e. Full-genome sequencing with a long-read method

e. Full-genome sequencing with a long-read method

Detecting differential expression means the comparison of two RNA-seq data sets. True or false?

true

Sequence assembly of short reads is more efficient because of the lower error rates. True or false?

false

RNA-seq can reveal alternative splicing for transcripts of a single gene because some of the reads will span across two exons, comfirming each splicing event. True or false?

true

a gene has six identified exons (1 to 6) in Ensembl and other databases. After mapping reads from RNA-seq you get the following numbers of reads which connect different exons, identifying splicing events. Any other non-consecutive exon combinations not shown in the table were not observed.

Exon-joining reads	number
1-2	13
1-3	10
2-3	0
2-4	12
3-4	14
4-5	22
5-6	28

Which of the following statements would be supported by the data? Choose one or more

a. The transcript containing all the exons 1 to 6 is the most common one

b. Exon 2 is skipped in approximately half of the transcripts

c. Exons 2 and 3 appear to be mutually exclusive alternative exons

d. There is evidence of at least three different transcripts

b. Exon 2 is skipped in approximately half of the transcripts

c. Exons 2 and 3 appear to be mutually exclusive alternative exons

Long sequencing reads have error rates of 5% to 15%. They are not usable because normally sequences are only accepted if the probability of error is less than 1%. True or false?

false

ChIP-seq is a method for studying which DNA sequences bind to a specific protein. You need to have an antibody to capture the protein of interest together with the DNA fragments it had bound. Then you release those fragments, sequence them and map the reads to the genome to discover which binding sites for that protein were occupied with the protein at the time of starting the experiment. True or false?

true

In RNAseq you only sequence the protein-coding regions of the genome. True or false?

false

If you want to study variants in the protein-coding sequences of one individual, the most appropriate experiment would be:

a. RNA-seq

b. Exome sequencing

c. CHIP-seq

d. Full-genome sequencing

b. Exome sequencing

There are no sequencing methods which could give information of modifications of the DNA bases, such as methylation. True or false?

false

After the reads from an RNA-seq experiment have been mapped (aligned) to the genome, the relative expression levels of genes can be inferred by comparing the amounts of reads aligned at the regions corresponding to the exons of each gene. True or false?

true

In comparison of RNA-seq data sets it is advisable to divide raw read numbers for each gene by total number of mapped reads to make the numbers more directly comparable between experiments. True or false?

true

A final result of many sequencing studies is a gene list, which could be genes overexpressed in cancer tissue compared to normal tissue of the same type in the same individual, or perhaps a list of all genes with a binding site for two specific transcription factors in their promoter region (regulatory region). In order to find what there is in common between all or some of the genes in the list, people often look for enrichment of some properties in the list. Enrichment means that you find that property more frequently within the list than in all genes. Choose any true statments regarding these enrichment analyses from the list below (one or more options)

a. a popular source of biological terms in enrichment anaysis is GO, Gene Ontology, which also contains hierarchical relationships between terms

b. Even if it is common to use the list of all human genes as the backround gene set against which the enrichment is calculated, this is not always the best choice. For example, if you compared a muscle tumour against normal muscle, using all genes as backround could show many normal muscle functions a enriched.

c. It is important to look at not only enrichment, but significant enrichment after a statistical analysis, such as Fisher’s Exact Test

d. It is common to analyze enrichment of biological terms attacted to genes and proteins. You can useany set of terms which have been used for consistent and comprehensive annotation of genes and/or proteins.

a. a popular source of biological terms in enrichment anaysis is GO, Gene Ontology, which also contains hierarchical relationships between terms

b. Even if it is common to use the list of all human genes as the backround gene set against which the enrichment is calculated, this is not always the best choice. For example, if you compared a muscle tumour against normal muscle, using all genes as backround could show many normal muscle functions a enriched.

c. It is important to look at not only enrichment, but significant enrichment after a statistical analysis, such as Fisher’s Exact Test

d. It is common to analyze enrichment of biological terms attacted to genes and proteins. You can useany set of terms which have been used for consistent and comprehensive annotation of genes and/or proteins.

Paired-end reads are a form of data you can get from Illumina short-read technology. The previous statement is correct, you only need to evaluate the truth of what follows. This means that you get the same sequence inferred from both strands of DNA so that the combination of the paired reads gives a more accurate sequence for that read. True or false?

false

Base calling means comparing your sequences to the reference genome to identify variant positions. True or false?

false

After variant calling you will know which positions in themapped sequence are the same as in the reference genome, different from the reference sequence, or have an approximately 50/50 mix of the reference allele and the alternative allele. It is impossible that there would be two different alternative alleles because we only have two copies of each gene. Do not take into account trisomic individuals or other copy number variants when deciding your answer. True or false?

false

In ChIP-seq you isolate a DNA-binding protein of interest with an antibody and then sequence the protein. True or false?

false

SMRT sequencing a.k.a. PacBio sequencing is one of the long-read methods. The previous statement is correct, you only need to evaluate what follows. Each read is derived from a single DNA molecule as it is being copied by a DNA polymerase. True or false?

true

Short-read sequencing methods require amplification of DNA fragments to create local clusters of copies of a single fragment. In sequencing each copy is intended to give the same signal synchronously in each sequencing round. As sequencing proceeds, the signal quality deteriorates as some of the copies are getting out of sync, and this puts an upper limit on how many rounds it is reasonable to sequence. True or false?

true

If you want to study gene expression levels, the most appropriate experiment would be:

a. RNA-seq

b. miRNA-seq

c. ChIP-seq

d. Genotyping

a. RNA-seq

After exome sequencing and mapping, the results for one particular position was 34 reads of C and 32 reads of T. This would lead into either proline or leucine in the protein sequence. What are your conclusions of the result? Choose one or more of the alternatives below.

a. The individual is a carrier of a genetic disease

b. C must be the reference allele because it shows more reads

c. The individual is heterozygous and propably expresses two different versions of the corresponding protein

d. This cannot be so, somtething must have gone wrong in sequencing or sequence assembly

c. The individual is heterozygous and propably expresses two different versions of the corresponding protein