各位同學(xué)，大家好，今天我們來(lái)分享一篇新的10X單細(xì)胞和10X空間轉(zhuǎn)錄組聯(lián)合分析文章贬蛙，文章在這里Spatiotemporal single-cell RNA sequencing 1 of developing hearts reveals interplay between cellular differentiation and morphogenesis,里面有很多經(jīng)典的10X單細(xì)胞和10X空間轉(zhuǎn)錄組的分析方法芽淡，今天我們的任務(wù)就是來(lái)參透這篇文章。
一嫂拴、ABSTRACT
Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about cellular interactions and tissue morphology（單細(xì)胞無(wú)法提供細(xì)胞的空間位置信息和組織形態(tài)學(xué)信息，這個(gè)已經(jīng)是單細(xì)胞很明顯的劣勢(shì)）贮喧，Here, we combined single-cell and spatial transcriptomics with new algorithms for data integration to study the early development of the chicken heart.（單細(xì)胞空間聯(lián)合來(lái)分析雞心臟的發(fā)育過(guò)程筒狠，這個(gè)很新穎，別說(shuō)單細(xì)胞空間聯(lián)合的文章箱沦，光單細(xì)胞的文章研究特殊物種的都很少）辩恼，We collected data from four key ventricular（心室的） development stages, ranging from the early chamber formation stage to the late four chambered stage.（取樣，相對(duì)很好取谓形，雞嘛运挫，比人的樣本要容易），We created an atlas of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions revealed the intricate interplay between cellular differentiation and morphogenesis in cardiac cellular lineages（得到的結(jié)果套耕，跟預(yù)想差不多）谁帕。Using spatially resolved expression analysis, we identified anatomically restricted gene expression programs. Last, we discovered a stage dependent role for the small secreted peptide, thymosin beta-4, in the coordination of multi lineage cellular populations. Overall, our study identifies key stage-specific regulatory programs that govern cardiac development.（看來(lái)細(xì)胞的空間位置，那是相當(dāng)重要）冯袍。
二匈挖、INTRODUCTION
這部分主要說(shuō)了一下幾方面的內(nèi)容
1、運(yùn)用10X單細(xì)胞空間兩種技術(shù)康愤，we combined spatially resolved RNA-seq with high throughput scRNA-seq to study the spatiotemporal interactions and regulatory programs that drive fetal development of the chicken heart儡循。（時(shí)空分析，這個(gè)看過(guò)我文章分析的同學(xué)應(yīng)該不陌生）征冷。Current spatial transcriptomics approaches lack single-cell resolution, which we addressed here using new approaches to integrate high throughput spatial and single-cell transcriptomic data.（兩種分析方法強(qiáng)強(qiáng)聯(lián)合）择膝。
2、為什么選擇雞的心臟樣本检激。the chick heart anatomy resembles the anatomy of the human heart more closely than other non-mammalian vertebrate model organisms（雞的心臟結(jié)構(gòu)跟人的很接近）肴捉。取雞心臟的樣本來(lái)運(yùn)用單細(xì)胞和空間技術(shù)。
3叔收、兩種技術(shù)聯(lián)合分析的效果齿穗。As we demonstrate here, the combination of single-cell and spatial transcriptomics uniquely enables to unravel cellular interactions that drive cardiogenesis.The data enabled us to reconstruct a high-resolution, spatially resolved gene expression atlas of epi-, endo-, and myocardial developmental lineages within cardiac tissue.We used the cell-type predictions to construct proximity maps revealing novel cellular interactions。我們注意一點(diǎn)饺律，We furthermore constructed a similarity map between single-cell and spatial transcriptomes, which enabled us to spatially map lineage-associated differentiation trajectories within the tissue.（單細(xì)胞軌跡分析在空間上的反映窃页，這個(gè)據(jù)我所知，應(yīng)該是首次）。
三脖卖、Result
（1）Spatially resolved single-cell transcriptomics atlas of developing fetal chicken hearts
這部分結(jié)果主要講了兩種技術(shù)的聯(lián)合運(yùn)用乒省。To study the complex interplay between differentiation and morphogenesis during cardiac development, we combined single-cell and spatial transcriptomics.看一下技術(shù)路線
不同時(shí)間點(diǎn)取樣，同時(shí)進(jìn)行10X單細(xì)胞和10X空間轉(zhuǎn)錄組技術(shù)畦木，然后單細(xì)胞空間的聯(lián)合分析作儿，最后是一些個(gè)性化，包括細(xì)胞之間的相互作用馋劈，軌跡分析等等。
spatial transcriptomes collected at the same developmental stage were strongly correlated（Pearson correlation; R > 0.98）晾嘶。同一時(shí)間段的單細(xì)胞和空間樣本具有極強(qiáng)的關(guān)聯(lián)性妓雾。To analyze single-cell transcriptomes, we filtered and preprocessed the data, performed batch correction using scanorama（單細(xì)胞樣本之間的批次去除采用了scanorama的方法，問(wèn)一下大家這個(gè)方法是哪個(gè)軟件的垒迂？知道的寫在評(píng)論區(qū)哈械姻，作者是知道的），我們看一下單細(xì)胞的分析結(jié)果机断。
雞心臟的細(xì)胞定義楷拳，相信非常的難，我們國(guó)內(nèi)的學(xué)者大部分做細(xì)胞定義完全沒(méi)有系統(tǒng)吏奸，可能這就是國(guó)內(nèi)外科研的差距吧欢揖，關(guān)鍵這細(xì)胞定義還是用marker gene定義的，這個(gè)定義過(guò)程及科研素質(zhì)奋蔚，令人敬佩她混。
接下來(lái)是空間數(shù)據(jù)的分析，populations, the spatial transcriptomics data was integrated with the scRNA-seq data using Seurat-v3 anchor-based integration（Seurat的聯(lián)合方法泊碑，這個(gè)方法我之前介紹過(guò)坤按，文章在10X空間轉(zhuǎn)錄組和10X單細(xì)胞數(shù)據(jù)聯(lián)合分析方法匯總），
In order to understand the spatial organization of cell types in broad anatomical regions, spots were labeled as cell types with maximum prediction score and visualized on H&E stained images of respective stages（每個(gè)SPOT定義成聯(lián)合分析中分?jǐn)?shù)最大的那種細(xì)胞類型馒过，如下圖)臭脓。
Cell-type prediction scores for spatial transcriptomes were further used to estimate the abundance of pairs of specific cell types（細(xì)胞共定位），As proximity is a necessity for physical interactions between two or more cells, these cell-type proximity maps can be used to guide the discovery of interactions between cell types from the same or different lineages.
（這個(gè)地方也是一個(gè)研究的重點(diǎn)腹忽，細(xì)胞之間的空間位置結(jié)果會(huì)體現(xiàn)出細(xì)胞之間的相互作用）来累。如下圖：細(xì)胞之間的定位關(guān)系
We found a significant number of cardiomyocytes colocalized with myocardial progenitor cells and precursor cells in all stages, as expected.（細(xì)胞之間的空間位置關(guān)系）。當(dāng)然窘奏，不同階段的細(xì)胞定位關(guān)系有所不同佃扼。
（2）Spatially resolved cardiac lineage analysis
Ventricular development in fetal hearts involves regulatory interactions and the coordinated
migration of cells from multiple lineages to form a fully developed four-chambered heart.（任何組織的發(fā)育都有時(shí)空的關(guān)系），
We first gathered and reclustered single-cell transcriptomes from the epicardial, endocardial, and myocardial lineages, and then reconstructed differentiation trajectories for these three lineages.（相同細(xì)胞類型的再分群和軌跡分析）蔼夜，We used PHATE (Potential of Heat-diffusion for Affinity-based Transition Embedding) to visualize differentiation trajectories because of its ability to learn and conserve local and global structure in low dimensional space（PHATE方法我之前分享過(guò)兼耀，這個(gè)方法目前來(lái)講降維可視化效果最好，文章在這里10X單細(xì)胞降維分析之PHATE）,PHATE1作為發(fā)育軌跡點(diǎn)，如下圖：
我們來(lái)看一下主要的分析結(jié)果
當(dāng)然這個(gè)圖得到了很多有意義的結(jié)果瘤运，但是這里作為信息分析人員窍霞，有以下分析點(diǎn)需要注意：
（1）、相同細(xì)胞類型的再分群拯坟。
（2）但金、PHATE降維可視化
（3）、單細(xì)胞空間聯(lián)合展示不同階段的細(xì)胞類型在空間上的位置郁季。
其中冷溃，作者也用了monocle2進(jìn)行軌跡分析。
（3）Spatiotemporally resolved local cellular heterogeneity in developing cardiac tissue
空間數(shù)據(jù)聚類梦裂，To examine transcriptional differences within the fetal cardiac ventricles,we performed unsupervised clustering of spatial RNA-seq spots and labeled the clusters by anatomical region based on their location in the tissue似枕。（注意看這里，空間聚類的注釋采用的是region年柠，這個(gè)地方一定要注意哈凿歼。）Using this analysis, we identified distinct
spatial clusters derived from ventricles, atria, valves, and the outflow tract but also distinct layers of ventricular regions including epicardium, compact and trabecular myocardium regions, and endocardium。
我們稍微結(jié)合一下上面的結(jié)果冗恨，第一部分的結(jié)果是利用單細(xì)胞的數(shù)據(jù)進(jìn)行空間信息的注釋答憔，而這里的結(jié)果卻是空間單獨(dú)聚類，進(jìn)行結(jié)構(gòu)注釋掀抹，We used cell type prediction scores for spatial transcriptomes as a proxy for cellular composition and analyzed temporal changes in local cellular composition for the major anatomical regions within ventricular tissue（這個(gè)地方很有意思虐拓，同一區(qū)域分析細(xì)胞類型成分的時(shí)間關(guān)系，值得注意傲武，是一個(gè)新的思路）侯嘀。
這個(gè)地方主要體現(xiàn)具體區(qū)域的細(xì)胞異質(zhì)性（空間marker，SeuratV3尋找空間高變基因）谱轨。
（4）A collective cell type and stage dependent role for thymosin beta-4
第四部分的結(jié)果我們就不分享了戒幔，大家自己看一看，
某一種細(xì)胞類型的詳細(xì)分析土童。
四诗茎、METHOD，這里我們關(guān)注一下幾種分析方法
（1）献汗、單細(xì)胞數(shù)據(jù)的處理
Scanorama去除批次效應(yīng)（什么軟件敢订？知道的寫在評(píng)論區(qū)里），單細(xì)胞分析軟件是SeuratV3罢吃。
（2）楚午、空間轉(zhuǎn)錄組的數(shù)據(jù)處理，也是SeuratV3尿招，包括單細(xì)胞空間聯(lián)合
（3）矾柜、軌跡分析
降維可視化 PHATE阱驾，同時(shí)進(jìn)行monocle2分析。
生活很好怪蔑，有你更好
