Table_6_Next Generation DNA-Seq and Differential RNA-Seq Allow Re-annotation of the Pyrococcus furiosus DSM 3638 Genome and Provide Insights Into Archaeal Antisense Transcription.XLSX

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old “lab culture” using Nanopore sequencing confirmed the overall stability of the P. furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.

Pyrococcus furiosus DSM 3638 作为一株嗜热古菌的模式生物，其最佳生长温度接近 100°C。该菌株的基因组测序工作约在 18 年前完成。然而，一些研究指出，与 Pyrococcus 其他物种相比，P. furiosus DSM 3638 的基因组更容易发生基因重排。因此，我们采用第三代测序技术对该基因组进行了重测序。新的从头组装基因组大小为 1,889,914 碱基对，与已发表的序列具有较高的序列一致性。然而，检测到两个主要差异：（1）由于近期描述的缺失，基因组较 NCBI 参考基因组小 18,342 碱基对。（2）PF0349 和 PF0388 之间的区域可能由于原始序列的组装问题而发生倒置。此外，还识别出大量微小的变异，包括单核苷酸交换、缺失或插入。新序列中的插入序列（IS）元件总数也从 30 个减少到 24 个。使用 Nanopore 测序对 2 年前的“实验室培养物”进行重测序，证实了 P. furiosus DSM 3638 基因组在正常实验室条件下即便未采取特殊保护措施也表现出整体的稳定性。为提升基因组注释，将更新的 DNA 序列与 RNA 测序方法相结合。在此，将来自八个不同生长条件的 RNA 池合，以增加检测到的转录本数量。此外，采用差异 RNA-Seq 方法来识别转录起始位点（TSS）。总计检测并分类出 2515 个 TSS，其中 834 个为初级（pTSS），797 个为反义（aTSS），739 个为内部 TSS，以及 145 个次级 TSS。我们对上游区域的解析揭示了高度保守的古菌启动子结构。通过调查 pTSS 和 aTSS 之间的距离，发现了大量反义转录本，这可能是由于来自同一 TATA 盒的双向转录所致。反义转录本生成的这种机制可以通过体外转录实验进一步验证。我们推测双向转录产生非功能性反义 RNA，且由于 TATA 元素的构型和 TATA 结合蛋白的对称结构，这种现象在古菌中普遍存在。