Development of robust analytical pipelines for the analysis of microbial community data from clinical samples

囊性纤维化是一种遗传性疾病，在英国约有9000人受到影响。这是一种隐性疾病，意味着父母双方都必须携带有缺陷的基因副本，孩子才会受到影响。大约每25人中就有1人携带这种复制品。囊性纤维化大大缩短了患者的寿命，几乎一半的人活不过30多岁。囊性纤维化对身体的许多部位都有负面影响，但它对肺部的影响尤其大。基因缺陷的结果是，厚厚的分泌物不能有效地从肺部清除，导致呼吸道充血和受损。这种充血导致频繁和严重的恶化，有时需要住院和治疗。我们认为，病情恶化往往是感染造成的，感染可能是由病毒和细菌引起的。发现的最常见的细菌之一是铜绿假单胞菌。微生物学家通过将样本放到平板上并识别在可见菌落中生长的细菌来诊断假单胞菌。假单胞菌通常用抗生素治疗。可能还会发现其他细菌，包括链球菌和葡萄球菌。但是，在寻找病情恶化的原因时，我们仅限于找到我们已知的病原体，这些病原体生长在我们使用的培养板上。可能存在其他我们看不见的细菌，因为它们不容易生长。有时，这些细菌可能是一个原因。然而，与人类肠道相似的是，细菌可能有“好”和“坏”之分。众所周知，某些类型的细菌可以防止其他类型的细菌感染，因此它们可能有助于防止病情恶化。一项新技术利用了分子条形码的概念，它可以从细菌细胞中的DNA片段中识别细菌物种。被称为高通量测序仪的新仪器允许从许多样本中轻松且廉价地读取这些条形码。这项技术为我们提供了一份特定样本中细菌种类的“部件清单”，并大致了解了它们出现的频率。通过阅读囊性纤维化患者的这一部分列表--当他们很好、当他们病得很重、当他们正在康复时，我们或许能够说出这些看不见的细菌对这种疾病的相对贡献。例如，一种特定物种的数量增加或减少可能与恢复有关，这给了我们一个潜在的治疗目标。我们也很感兴趣地观察患者最终是如何被特定的、常见的细菌定植的。例如，在我们当地的患者中，约有30%的人感染了一种特殊类型的假单胞菌，称为米德兰1号。然而，人们对如此多的患者最终感染同一菌株的原因知之甚少。我们现在能够对细菌细胞(基因组)中的所有DNA进行排序，这给出了关于它是如何进化的非常高分辨率的观点。通过比较不同患者的菌株基因组，我们可以帮助确定患者是否相互感染相同的菌株，或者这些菌株是否非常不同，来自许多不同的来源。我们还可以看到假单胞菌在患者肺部时是如何进化的。之前的研究表明，假单胞菌能适应特定的环境，这可能会给我们提供线索，为什么患有相同囊性纤维化突变的人有不同的病程，最终比其他人有更多的住院或病情恶化。我们正在使用的技术非常新，在它可以成为常规临床测试之前，它存在一些困难。一个问题是，这些机器会产生大量的测序“噪音”，看起来就像是存在但并不存在的物种。我想开发生物信息学方法，试图提高这些技术的可靠性，并产生对临床医生有用的信息。我们预计这些技术将在未来五年内进入临床。

Cystic fibrosis is an inherited disease which affects around 9,000 people in the UK. It is a recessive disorder, meaning that both parents have to carry a faulty copy of a gene for a child to be affected. Approximately 1 in 25 people carry this copy. Cystic fibrosis dramatically shortens the life of those affected and almost half do not live beyond their 30s. Cystic fibrosis has a negative effect on many parts of the body, but it particularly affects the lungs. The result of the defective gene is that thick secretions cannot be effectively cleared from the lungs, resulting in the airways becoming congested and damaged.This congestion results in frequent and severe exacerbations, sometimes requiring admission to hospital and treatment. We think exacerbations are often a result of infections, which may be caused by viruses and bacteria. One of the most common bacteria found is Pseudomonas aeruginosa. Microbiologists diagnose Pseudomonas by putting samples of sample onto plates and identifying the bacteria that grow in visible colonies. Often Pseudomonas is treated by antibiotics. Other bacteria may be found including Streptococcus and Staphylococcus.However when looking for causes of exacerbations we are limited to finding pathogens that we know about, and which grow on the types of culture plates we use. It may be that other bacteria are present that we can't see because they do not grow easily. Sometimes those bacteria may be a cause. However, in a similar way to the human gut it may be that there are "good" and "bad" bacteria. It is known that certain types of bacteria can prevent other types infecting and therefore they may help protect against exacerbations.A new technology utilises the idea of molecular barcodes which identify bacterial species from fragments of DNA in their cell. New instruments termed high-throughput sequencers permit these barcodes to be read from many samples easily and cheaply. This technology gives us a "parts list" of the bacterial species in a particular sample, and a rough idea of how frequently they occur. By reading this parts lists from patients with cystic fibrosis - when they are well, when they are very sick and when they are recovering, we may be able to tell the relative contribution of these unseen bacteria to the condition. For example a particular species increasing or reducing in abundance may be associated with recovery, giving us a potential therapeutic target.We are also interesting in seeing how patients end up being colonised with particular, commonly seen bacteria. For example in our local patients, about 30% have a particular type of Pseudomonas infection called Midlands 1. However, very little is known about how it is that so many patients end up being infected by the same strain. We are now able to sequence all the DNA in a bacterial cell (the genome) which gives very high resolution view of how it has evolved. By comparing genomes of strains from different patients, we can help determine whether patients are infecting each other with the same strain, or whether the strains are quite different and come from many different sources. We can also see how the Pseudomonas evolves whilst it is in a patients lungs. Previous studies have shown that the Pseudomonas adapts to the specific environment, which may give us clues as to why people with the same cystic fibrosis mutation have different courses and end up with more hospital admissions or exacerbations than others.The technology we are using is very new, and there are a number of difficulties with it before it can be a routine clinical test. One problem is that the machines generate plenty of sequencing "noise" which may look like species are present that aren't. I want to develop bioinformatics methods that try and increase the reliability of these techniques and generate information that would be useful for clinicians. We expect these techniques to enter the clinic within the next five years.