Hinsu Ankit, Dumadiya Ashvin, Joshi Anjali, Kotadiya Rohitkumar, Andharia Kavan, Koringa Prakash, Kothari Ramesh
Department of Biosciences, Saurashtra University, Rajkot, India.
Department of Animal Biotechnology, Anand Agricultural University, Anand, India.
PeerJ. 2021 Sep 1;9:e12035. doi: 10.7717/peerj.12035. eCollection 2021.
Sequencing driven metagenomics studies have been instrumental in various aspects of microbiology including identification of newer taxa. While this culture-independent approach has its own merits and demerits, several studies have focussed on comparing it with traditional culture-dependent (CD) approach. However, most of these comparative studies rely on Sanger sequencing of complete 16S rRNA gene from pure culture colonies to determine the culturable bacterial diversity. This approach undercounts culturable diversity as only fewer isolates are selected, sequenced, and identified.
In this study, we have used an Illumina based partial 16S sequencing to identify all the microbes growing on the media and directly comparing with its culture-independent (CI) counterpart. Eight different media were used to target different organisms from soil. Diversity on these media were compared with their CI counterpart. The NGS data was analysed using DADA2 to provide more resolution to the data.
In line with studies of similar nature, current study presented higher bacterial diversity in CI approach. However, the current study reflected that a greater number of sequence variants were missed out in CI approach as compared to number of sequence variants shared with CD approach. We observed around 322 (5.98%) ASVs (Amplicon Sequence Variants) exclusively present in CD samples while, 234 (4.35%) ASVs were shared between both approaches. Most of these 322 CD exclusive ASVs were classified as family and genus, with several ASVs annotated at the species level as well, and these organisms are more commonly observed in soil and were also detected in CI approach. Furthermore, 22 genera were exclusively detected in CD samples, most of which were reported from soil and water.
测序驱动的宏基因组学研究在微生物学的各个方面都发挥了重要作用，包括新分类群的鉴定。虽然这种不依赖培养的方法有其自身的优缺点，但已有多项研究致力于将其与传统的依赖培养（CD）方法进行比较。然而，这些比较研究大多依赖于对纯培养菌落中完整的16S rRNA基因进行桑格测序，以确定可培养细菌的多样性。由于仅选择、测序和鉴定了较少的分离株，这种方法低估了可培养的多样性。
在本研究中，我们使用基于Illumina的16S部分测序来鉴定在培养基上生长的所有微生物，并直接与其不依赖培养（CI）的对应物进行比较。使用八种不同的培养基来靶向土壤中的不同生物体。将这些培养基上的多样性与其CI对应物进行比较。使用DADA2分析NGS数据，以提高数据的分辨率。
与类似性质的研究一致，本研究表明CI方法中的细菌多样性更高。然而，当前研究表明，与CD方法共享的序列变体数量相比，CI方法遗漏了更多的序列变体。我们观察到约322个（5.98%）扩增子序列变体（ASVs）仅存在于CD样本中，而两种方法之间共享234个（4.35%）ASVs。这322个CD独有的ASVs大多被分类到科和属，也有一些ASVs在物种水平上被注释，这些生物体在土壤中更常见，在CI方法中也被检测到。此外，在CD样本中仅检测到22个属，其中大多数是从土壤和水中报道的。
