Question

1. Comparison of the Neanderthal genome to the modern human and chimpanzee genomes led to the conclusion that C->T and G -> A transition mutations were over-represented in the branch of the tree that led to Neanderthals. How could you tell whether a mutation was “over-represented”? Compared to what? Explain the specific methods and comparisons that were used to reach this conclusion, and how the results were interpreted. If a particular mutation was over-represented in ancient DNAs, then what sorts of molecular mechanism(s) you suspect? In other words, when did these excess C->T and G -> A transitions occur – (i) during reproduction in the lives of Neanderthals, or (ii) during post-mortem changes to their tissues over the centuries? Why?

Answer 1

Kindly find below the answers with complete explanation to each of it.

Modern humans are distinguished from archaic humans by changes in gene sequences. The understanding of this can be drawn by examining genomic DNA of an archaic hominid (especially Neanderthals) that diverged from the lineage before emergence of contemporary humans. The anatomical similarity to modern humans and for the ease of availability of remains from which DNA can be extracted, make Neanderthals the favourite choice to work with.

Challenges faced while working with Neanderthal DNA can be broadly classified as damage-induced mutations or contamination with modern human DNA, both of which reduced the authenticity to use Neanderthal Genome for evolutionary analysis purposes. Following are the different challenges faced:

DNA from fossil that are 100-1000years old. DNA is most cases have been damaged and found not to be useful. Completely worn out/ degraded DNA will not appear at all.

DNA if present may be in small fragments of 50-100bp dues to damage occurred over time and thus correct estimate of genes may not be available.

Most of the recovered and extracted DNA have been contaminated with microbes that have been growing and decaying in and around the area where from the remains are found. The contamination was confirmed by performing Sanger sequencing of Neanderthals DNA against Pleistocene animal, which revealed only 1.3% similarity and rest of the Neanderthal extracted DNA matched prokaryotic genomes.

Chances of contamination also arise during handling and extraction process, where modern human DNA may contaminate the available Neanderthal genome.

THUS, COMPARISON OF NEANDERTHAL WAS MADE TO BOTH MODERN HUMANS AS WELL AS ANCIENT PLEISTOCENE ANIMAL (LIKE CAVE BEAR).

Neanderthal convergence and mutation:

When compared to humans and chimpanzees, a 65kB sequence of the Neanderthal genome showed a distinctive “specific substitution” pattern biased towards C to T and A to G transition. This may be suggestive of errors due to cytosine deamination. It has been found that cytosine deamination causes the majority of damage-induced mutations.

When comparing human genome to Neanderthal, there are chances of having an overestimation of Neanderthal substitution, because the probability of mistaking error and substitutions are very high, and will be considered together, often. A corollary of this will be underestimating modern human genome, at places where Neanderthal sequences may by chance match the human state.

The possibility of errors or polymorphism can be screened out by increasing the sequence coverage. The reason being, deamination events do not occur at high frequency at the very same sites in multiple independent ancient DNA types. Cloning and sequencing multiple PCR products may help achieve this.

The CYTOSINE DEAMINATION IN THIS CASE OCCURS POST-MORTEM. This can be said because PCR and sequencing should ideally resolve bisulphite conversion, but in practical experience, C to T was obtained. This happens when unmethylated cytosine is converted to uracil and appears as thymine, following a PCR amplification reaction. Find attached the figure below for further clarity.

Techniques employed to obtain sequences:

PCR amplification of short mitochondrial sequences

Metagenomic approach using high throughput sequencing for genomic sequences – includes Sanger sequencing, high throughput Illumina (genome analyser) sequencing, Pyrosequencing

Advantage of the study: Used as a marker to study evolution of modern humans