NEW YORK – One of the inventors of the Mission Bio Tapestri single-cell analysis platform has adapted its workflow to do single-cell microbial sequencing.

纽约——Mission Bio Tapestri单细胞分析平台的发明者之一已调整其工作流程，以进行单细胞微生物测序。

Researchers led by Adam Abate of the University of California, San Franciso, have tinkered with the Tapestri chemistry to isolate individual bacteria and sequence their genomes. Easily Accessible Single-microbe sequencing (EASi-seq) adds a manual gel encapsulation and lysis step in front of the standard multi-day Tapestri droplet isolation and DNA amplification workflow.

加州大学旧金山分校的亚当·阿贝特领导的研究人员改进了Tapestri化学技术，以分离单个细菌并对其基因组进行测序。易获取的单微生物测序（EASi-seq）在标准的多天Tapestri液滴分离和DNA扩增流程之前，增加了一个手动的凝胶封装和裂解步骤。

It also changes the first step on the instrument to tagmentation instead of lysis. .

它还改变了仪器上的第一步，将裂解改为标记。

'Our challenge was to do something upfront [of the Tapestri workflow] while keeping it simple,' Abate said. Tapestri offers the ability to perform two chemical reactions onboard, but Abate's lab needed three. 'We wanted it to be something anyone can do. Usually, you do it with [custom] microfluidics, but that defeats the whole purpose,' which was to make it accessible to more labs, he said, as custom microfluidics require a high degree of skill to operate..

“我们的挑战是在Tapestri工作流程前期做一些事情，同时保持简单，”阿贝特说。Tapestri能够执行两个板载化学反应，但阿贝特的实验室需要三个。“我们希望这是任何人都可以做到的事。通常情况下，你会用[定制]微流控技术来实现，但这违背了整个目的，”他说，定制微流控技术需要很高的操作技能，而目的是让更多的实验室能够使用这项技术。

Runs result in tens of thousands of genomes and can be used to generate large atlases of microbial communities. The researchers published a proof-of-concept paper on their method earlier this month in

运行结果产生了数以万计的基因组，可以用来生成大型微生物群落图谱。研究人员本月初发表了关于他们方法的概念验证论文。

Advanced Science

先进科学

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。

'It's like single-cell on steroids,' said Rohan Sachdeva, a microbe researcher at the Innovative Genomics Institute and the University of California, Berkeley. '

“这就像打了类固醇的单细胞，”加州大学伯克利分校创新基因组学研究所的微生物研究员罗汉·萨切蒂瓦说。

It overcomes issues with scale and cost you have to deal with when using traditional single-cell bacterial genomics methods.

它克服了使用传统单细胞细菌基因组学方法时必须面对的规模和成本问题。

' Sachdeva has sent samples to Abate's lab to be assayed by EASi-seq but does not have a Tapestri so has not run it himself. '

萨切德瓦已将样品送到阿巴特的实验室通过EASi-seq进行分析，但他自己没有Tapestri，所以还没有亲自运行。

Being able to do that in our own group is where we want to be, but it's not exactly there yet. I think you could recreate all this, but it's not like you can go out and buy a kit, which is where I would want to be.'

能够在我们自己的团队中做到这一点是我们想要达到的目标，但目前还没有完全实现。我认为你可以重新创建所有这些，但这并不意味着你可以出去买一套现成的工具包，这才是我想要达到的状态。

EASi-seq builds on a previous method for single-cell microbial sequencing developed by Abate's lab, dubbed

EASi-seq 建立在 Abate 实验室之前开发的单细胞微生物测序方法的基础上，该方法被称为

SiC-seq

碳化硅序列

(single-cell genomic sequencing), which was licensed to Mission Bio.

（单细胞基因组测序），该技术已授权给Mission Bio。

Single-cell genomics can be as powerful for microbial as it has been for mammalian biology. 'In many ways, the microbial world is far more diverse than the mammalian cell world,' Abate said. 'That diversity actually is a major challenge in just characterizing the types of microbes that are present in any given system.'.

单细胞基因组学对微生物的研究可以像对哺乳动物生物学一样强大。阿贝特说：“在很多方面，微生物世界比哺乳动物细胞世界更加多样化。” “这种多样性实际上是在描述任何给定系统中存在的微生物类型时的一个主要挑战。”

SiC-seq is one of several methods developed over the last 20 years that have attempted to isolate and analyze individual microbes. For example, in 2022, David Weitz's lab at Harvard University came out with

SiC-seq 是过去 20 年中开发的几种试图分离和分析单个微生物的方法之一。例如，2022年，哈佛大学大卫·威茨的实验室推出了

Microbe-seq

微生物测序

, a droplet microfluidics-based method that amplifies genomes for sequencing. Researchers have also developed single-cell RNA sequencing methods, such as

，一种基于液滴微流控技术的方法，可扩增基因组以进行测序。研究人员还开发了单细胞 RNA 测序方法，例如

smRandom-seq

sm随机序列

from Yongcheng Wang's lab at China's Zhejiang University School of Medicine.

来自中国浙江大学医学院王永成实验室。

Some companies have even sprung up in the single-cell shotgun sequencing space, including

一些公司甚至在单细胞鸟枪法测序领域崭露头角，包括

BitBiome

比特生物群系

and

和

Atrandi Biosciences

安特兰迪生物科学

.

。

To improve throughput, many methods use custom microfluidics to help isolate cells and deliver the reagents needed to prepare sequencing libraries. However, this creates a high barrier to adoption. ' You have to fab your own consumables and reagents,' Abate said. '[I]t’s very difficult to do reproducibly.

为了提高通量，许多方法使用定制的微流控技术来帮助分离细胞并提供制备测序文库所需的试剂。然而，这造成了很高的应用障碍。阿贝特说：“你必须自己制作耗材和试剂，要做到可重复性非常困难。”

They need to be exactly right or they just give you garbage.'.

它们必须完全正确，否则只会给你垃圾数据。

Having designed the technology that Tapestri is based on, Abate thought that it could help with reproducibility. 'We wanted to show that the platform had all of the power necessary for microbe sequencing and that it just required a bit of tweaking,' he said. 'It turned out to be quite a lot of tweaking.'.

阿贝特设计了Tapestri所基于的技术，他认为这将有助于提高实验的可重复性。“我们想证明这个平台具备微生物测序所需的所有功能，只需要稍作调整即可，”他表示，“结果发现需要进行相当多的调整。”

EASi-seq is the result of years of work to get the method right. Some single-cell instruments, such as 10x Genomics' Chromium controller, perform cell isolation and library prep in one step. Tapestri does two, however, Abate's team needed three. Moreover, they felt they needed to separate cell lysis from whole-genome amplification.

EASi-seq 是经过多年努力才得以完善的方法。一些单细胞仪器，例如 10x Genomics 的 Chromium 控制器，可以一次性完成细胞分离和文库制备。然而，Tapestri 需要两步，而 Abate 的团队则需要三步。此外，他们认为需要将细胞裂解与全基因组扩增分开进行。

To make matters more challenging, they needed a way to lyse many different kinds of bacteria, and Tapestri normally amplifies targeted regions of the mammalian genome, not a whole bacterial one. .

为了使问题更具挑战性，他们需要一种方法来裂解许多不同种类的细菌，而Tapestri通常扩增哺乳动物基因组的靶向区域，而不是整个细菌基因组。

Their solution was to encapsulate bacterial cells into a hydrogel to lyse them so that their contents stay together. It's a manual step where a technician uses a syringe to make droplets, then centrifuges them to separate them by size and select spheres of a certain diameter. It takes a few minutes to encapsulate the cells and a few hours to sort and extract them.

他们的解决方案是将细菌细胞包裹在水凝胶中，使其裂解，以便其内容物保持在一起。这是一个手动步骤，技术人员使用注射器制作液滴，然后通过离心按大小分离并选择特定直径的球体。封装细胞需要几分钟，而分选和提取它们则需要几个小时。

'You can produce far more encapsulated cells than you could ever sequence,' Abate said. The hydrogel spheres conveniently have similar physical properties to a mammalian cell, allowing them to flow through the Tapestri system. .

“你可以生成比你能够测序的多得多的封装细胞，”阿贝特说。水凝胶球体具有与哺乳动物细胞相似的物理特性，可以方便地流经Tapestri系统。

The method also changes the reagents delivered in each Tapestri step. Normally, Tapestri lyses cells in the first step, but EASi-seq does tagmentation to barcode the contents from the previously lysed cell.

该方法还会改变每个Tapestri步骤中交付的试剂。通常，Tapestri 在第一步裂解细胞，但 EASi-seq 会进行标签化，以对先前裂解细胞的内容物进行条形码标记。

To create a 10,000-cell atlas, consumables costs total approximately $1,500: about $100 for the microfluidic cartridge; $300 to $400 for reagents including enzymes, buffers, and barcodes; and about $1,000 for sequencing. Accounting for the labor and overhead to perform each step significantly increases costs, however, 'they typically scale more slowly with larger projects, allowing larger-scale studies to achieve economies of scale not feasible in smaller projects,' Abate said.

创建一个包含一万个细胞的图谱，耗材成本总计约为1500美元：微流控卡盒约100美元；包括酶、缓冲液和条形码在内的试剂为300至400美元；测序费用约为1000美元。然而，执行每一步所需的人工和间接费用显著增加了成本。阿贝特表示：“它们通常在大型项目中增长得更慢，从而使大规模研究能够实现小规模项目无法实现的规模经济。”

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How accessible the method will be remains to be seen. Abate, a Mission Bio cofounder, and several of his coauthors have applied for patents related to EASi-seq. Tapestri is commercially available but generally marketed towards cancer researchers, and Mission Bio's VP of Global Marketing, Vanee Pho-Conners, said the firm has 'no immediate commercial plans to disclose.' .

这种方法的可及性还有待观察。阿巴特，Mission Bio 的联合创始人，和几位合著者已经申请了与 EASi-seq 相关的专利。Tapestri 已经在市场上销售，但通常面向癌症研究人员推广，并且 Mission Bio 的全球营销副总裁范妮·普霍-康纳斯表示，公司“没有立即披露的商业计划”。

One reason Sachdeva is interested in EASi-seq is to learn more about bacterial plasmids. He's trying to deliver CRISPR-Cas9 genome editing systems to the cow microbiome in order to reduce methane emissions, and plasmids are a key part of that. However, standard metagenomic approaches make it hard to link plasmids to other elements of the bacterial genome.

萨切德瓦对EASi-seq感兴趣的原因之一是想更多地了解细菌质粒。他试图将CRISPR-Cas9基因编辑系统递送到牛的微生物组中，以减少甲烷排放，而质粒是其中的关键部分。然而，标准的宏基因组学方法很难将质粒与其他细菌基因组元素联系起来。

'.

。

It's like trying to put a puzzle back together where you didn't have the jigsaw ends to recreate the picture,' he said. 'You don't have the parts that click together to link a plasmid to a host.'

“这就像试图把一个没有拼图边缘的拼图重新拼凑起来，”他说。“你没有可以将质粒与宿主连接起来的部件。”

EASi-seq is not a whole-genome method, covering up to around 5 percent of each genome. 'However, even at this modest coverage, our barcoded single-cell approach provides critical information unavailable through conventional shotgun metagenomics,' Abate said. 'Because all reads from an individual cell are physically linked within their droplet, we can confidently assign genomic fragments, plasmids, and viral sequences to their specific host organism.

EASi-seq 并非一种全基因组方法，覆盖每个基因组的约5%。 “然而，即使在这种适度的覆盖范围内，我们带有条形码的单细胞方法也提供了传统鸟枪法宏基因组学无法获得的关键信息，”阿贝特说。 “由于来自单个细胞的所有读数都在其液滴内物理连接，我们可以确信地将基因组片段、质粒和病毒序列分配给其特定的宿主生物。”

This intrinsic linkage makes it possible to precisely resolve strain-level differences, track the movement of mobile genetic elements, and integrate single-cell genomes with bulk metagenomic data for greater accuracy. Thus, the significant advantage is not necessarily in the completeness of each genome, but in the rich relational insights provided by analyzing tens of thousands of single-cell genomes, even if [at] low coverage.'In its study, the Abate lab used EASi-seq to produce atlases of a human gut microbiome and a coastal sea water microbiome.

这种内在联系使得精确解析菌株水平的差异、追踪移动遗传元件的运动以及将单细胞基因组与宏基因组数据整合成为可能，从而提高准确性。因此，其显著优势不一定在于每个基因组的完整性，而在于通过分析数万个单细胞基因组所提供的丰富关联信息，即使覆盖度较低亦是如此。在研究中，阿贝特实验室使用EASi-seq技术生成了人类肠道微生物组和沿海海水微生物组的图谱。

But even he was unsure of how the field would use this capability. 'Single-cell microbial genomics is still a relatively new field, so precedents for using microbial atlases are limited,' he said..

但是，就连他自己也不确定这个领域会如何利用这种功能。他说：“单细胞微生物基因组学仍然是一个相对较新的领域，因此使用微生物图谱的先例有限。”

Still, the genomes could also serve as reference scaffolds for improving bulk metagenomic assemblies, Abate suggested. 'The physical linkage preserved by hydrogel barcoding reveals genuine biological associations — such as viral–host or microbe–parasite interactions — that conventional metagenomic sequencing would disrupt.

阿贝特提出，基因组还可以作为改进大规模宏基因组组装的参考框架。 “水凝胶条形码保留的物理连接揭示了真实的生物学关联，例如病毒与宿主或微生物与寄生虫之间的相互作用，而传统的宏基因组测序会破坏这些关联。”

Many additional applications, including studies of horizontal gene transfer and strain-level dynamics during infections, will likely emerge,' he said..

他说：“许多额外的应用，包括对感染期间水平基因转移和菌株水平动态的研究，可能会出现。”