9. Gene Duplications, Transposable Elements and Genome Size

Which statements regarding human pseudogenes are correct?

1) Probably, most pseudogenes have a function that has just not been discovered yet -> wrong, they are nonfunctional homologs (but there are also likely some few pseudogenes, that have a unknown function)

2) The exon-intron structure of a processed pseudogene and its functional homolog is essentially identical -> wrong, a processed pseudogene goes via an RNA-intermediate, so the intron-exon structure is not preserved

3) There are currently ~1000 pseudogenes annotated in the human genome -> wrong, there are 15k annotated

4) Most human pseudogenes contain just one mutation that disrupts the coding sequence -> wrong, if a pseudogene “sticks around” for million years -> it will have several mutations; most pseudogenes, if they arose a while ago, are totally “shredded”, only pseudogenes that arose very recently might have just 1 mutation

5) Most human pseudogenes are processed pseudogenes -> correct

Which statements regarding gene duplications are correct?

1) Susumo Ohno proposed in his famous book that gene duplications play a big role during evolution, but genome projects have shown that gene families are rare -> wrong, genome projects have shown that gene families are widespread

2) When an entire gene gets duplicated, it is most likely that the two paralogs acquire different or new functions -> wrong, “we are already good adapted -> if one makes a copy its very unlikely that it will make it better”; if a duplication happens a priori it’s unlikely that it will be good

3) When genes gets duplicated via a mRNA intermediate, the process is called retrotransposition -> correct

4) If two genes of two different species are called 1:1 orthologs, this means they have been generated by gene duplication -> wrong, they have been generated by species duplication; gene duplication is “for” Paralogs

5) When a paralog undergoes subfunctionalization after a gene duplication its sequence is expected to evolve neutrally -> wrong, = copy gets adapted to a new function -> no neutrally ofc, not like a pseudo; either not needed, then it evolves neutrally and becomes a pseudogene eventually or it acquires a new function and evolves adapted, very likely undergoes positive selection, optimizes and becomes eventually a gene

Which statements regarding (DNA-) Microarrays are correct?

1) Microarrays have been the major method in the last decade to quantify gene expression levels genome wide -> correct

2) Affymetrix is a major company selling microarrays -> correct

3) Biotin-labeled RNA is generated from the sample of interest and is then hybridized to the Affymetrix microarrays -> correct

4) Microarrays are the basic technology for third-generation sequencing -> wrong

5) Over a million different oligonucleotides can be synthesized on an Affymetrix microarray -> correct (newer versions up to 6 million)

Which statements regarding RNA-Seq (Illumina) are correct?

1) For quantitative RNA-seq, read lengths that allow mapping are sufficient -> correct

2) Reverse transcriptase is always needed to make RNA-Seq libraries -> correct

3) RNA-seq libraries should have as long as possible inserts so that full length cDNA can be sequenced -> wrong

4) A few thousand sequencing reads are usually sufficient to characterize genome-wide expression patterns -> wrong

5) To quantify gene expression levels by RNA-seq one usually assembles the short reads into contigs before mapping -> wrong

What is the correct order of these steps when generating an RNA-Seq library?

1) first-strand synthesis -> 4.

2) RNA fragmentation -> 3.

3) adding library barcodes by PCR -> 7.

4) total RNA isolation -> 1.

5) ligating adapters -> 6

6) second strand synthesis 5. 

7) polyA enrichment -> 2.