Table_5_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 被视为一株高温嗜热古菌的模式生物，其最适生长温度接近 100°C。该菌株的基因组大约在 18 年前完成测序。然而，一些研究指出，与 Pyrococcus 其他物种相比，P. furiosus DSM 3638 的基因组更易发生基因组重排。因此，我们采用第三代测序技术对该基因组进行了重新测序。新的从头组装基因组大小为 1,889,914 碱基对，与已发表的序列展现出高度的序列一致性。然而，检测到两个主要差异：（1）由于最近描述的缺失，基因组比 NCBI 参考基因组小 18,342 碱基对。（2）PF0349 与 PF0388 之间的区域发生了倒置，这很可能是因为原始序列的组装问题。此外，还发现了大量细微变异，包括单核苷酸交换、缺失或插入。新的序列中插入序列（IS）元素的总数也从 30 减少到 24。使用纳米孔测序对两年前的“实验室培养物”进行重新测序，证实了即使在常规实验室条件下，P. furiosus DSM 3638 的基因组整体稳定性也未受到影响，无需采取任何特殊保养措施。为了提高基因组注释，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 池合并，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）。总共检测到 2515 个 TSS，并将其分类为 834 个主要（pTSS）、797 个反义（aTSS）、739 个内部和 145 个二级 TSS。我们对上游区域的分析揭示了高度保守的古菌启动子结构。对 pTSS 与 aTSS 之间的距离进行探究，发现大量反义转录本，这是由于来自同一 TATA 盒的定向转录的结果。这种反义转录本产生的机制可以通过体外转录实验进一步证实。我们假设双向转录产生了非功能性反义 RNA，并且由于 TATA 元素的结构和 TATA 结合蛋白的对称结构，这种现象在古菌中普遍存在。