DNA extraction and sequencing

Following acquisition, sedaDNA samples are sent to aDNA-dedicated laboratories for aDNA extraction and sequencing. Next-generation sequencing (NGS) (also known as high-throughput sequencing (HTS)) refers to modern sequencing technologies which can perform the sequencing of millions of small fragments of DNA in parallel. In NGS procedures, the generation of aDNA sequence reads comprise 1) library preparation, 2) library amplification and 3) sequencing. Library preparation entails the ligation of specific adaptor oligos to fragments of the DNA to be sequenced. The DNA is fragmented and then repaired (at the 3’ and 5’ ends) to facilitate the ligation of adapters to the DNA. This is required for PCR amplification and the preparation of DNA fragments into a form that is compatible with the sequencing system used (e.g. the Illumina MiSeq or HiSeq). Despite the sensitivity of NGS sequencers, signal detection systems cannot measure signals originating from a single DNA molecule. Amplification of the sequencing library is therefore required to obtain sufficient sequence reads. Libraries are then sequenced on a high-throughput sequencing system (e.g. the Illumina HiSeq 2500). Because of the nature of the chemistry and amplification involved, Illumina sequencers tend to produce ‘short’ reads with a length of 36 to 150 bases. The Illumina sequencers are therefore perfect for aDNA, which typically comprises only 60 to 150 bp in length.