Review
Single cell sequencing:technique,application,and future development
Ruidong Xue ?Ruoyan Li ?Fan Bai
Received:3July 2014/Accepted:8August 2014/Published online:17December 2014óScience China Press and Springer-Verlag Berlin Heidelberg 2014
Abstract The progression of next generation sequencing is continuously changing the landscape of genomic,tran-scriptomic,and epigenomic studies.Particularly,advances in single cell manipulation and ampli?cation techniques bring sequencing technology to the single-cell level.Single cell genome sequencing allows us to study tumor evolu-tion,gamete genesis,somatic mosaicism at genome-wide level;single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel-opment,differentiation and reprogramming;single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies.Lots of attempts are still being made in other dimensions of sequencing.The increasing need for single cell sequencing requires the future techniques with the following features:(1)high accuracy and ?delity;(2)able to perform multiple omics analyses in one cell;(3)high degree of automation and standardized pipeline.These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories.The wide application of single cell sequencing techniques will substantially change biomedi-cal research in future.
Keywords Single cell isolation áGenetic
heterogeneity áSingle cell genome sequencing áSingle cell transcriptome sequencing áSingle cell methylome sequencing
Many biological experiments and assays are performed on cells in bulk,assuming that all cells of a particular ‘‘type’’are identical.However,recent evidence from single cell studies reveals that this assumption is not completely true [1,2].Individual cells within the same population may,on various aspects,differ dramatically,thus making different contributions to the health and function of the entire pop-ulation.Experimental approaches that only measure the population-level characteristics may average or dilute the vital differences among cells.For instance,the heteroge-neous nature of cancer cells makes it dif?cult and inaccu-rate,by bulk sequencing,to assess the genetic clonal architecture of tumors at genome-wide level.On the other hand,cells of a particular type,such as early embryos and unculturable microbes,are precious and scarce.As with next generation sequencing (NGS),the demand for a suf-?cient amount of DNA or RNA turns out to be a major obstacle for direct detection of diverse genomic,tran-scriptomic and epigenetic events in these cells.For both reasons,much attention has been paid to the development of single cell analysis methods.Recently,great progresses have been achieved in single cell sequencing,which stands out among all single cell analysis techniques.
The past decade witnessed the sustained efforts that have been made in developing sequencing techniques:First,the introduction of NGS with higher throughput and lower costs has revolutionized genomic studies [3].Gone was the time when enormous men and machines,supported by a super big budget,were put into work for years to sequence a human genome.Nowadays,the same task can be performed within a few days.What’s more,various ef?cient single cell ampli?cation methods have been introduced to bridging the gap between the trace amount of genetic materials obtainable from a single cell and the minimum sample quantity required for NGS.In addition,
R.Xue áR.Li áF.Bai (&)
Biodynamic Optical Imaging Centre (BIOPIC),College of Life Sciences,Peking University,Beijing 100871,China e-mail:fbai@https://www.doczj.com/doc/b717978452.html,
Sci.Bull.(2015)60(1):33–https://www.doczj.com/doc/b717978452.html,
DOI 10.1007/s11434-014-0634-6
the improvement of single cell isolation and manipulating methods,such as the ?uorescence-activated cell sorting (FACS),micromanipulation,and the use of micro?uidic devices,greatly enhance researchers’ability to handle a large number of single cells from diverse sources.Inte-grating these advancements together,single cell sequenc-ing achieves breakthroughs in numerous research areas,and ?nally draws great public attention.In particular,sin-gle cell sequencing is selected as the ‘‘Method of the Year for 2013’’by Nature Methods for its potential in under-standing life one cell at a time [4].The past several years have seen single cell sequencing ?nding applications in diverse research areas ranging from sequencing of microbes,haplotype analysis,early embryonic develop-ment,cancer research,prenatal genetic diagnosis to human genome mosaicism.In this review,we will focus on recent highlights of single cell sequencing techniques (Fig.1)and discuss the prospect of this blooming ?eld.
1Single cell isolation
The very ?rst challenge that single cell isolation meets is to pick the right target cells.Some studies focuses on single cell samples that can be picked up directly,for instance sperms and oocytes,while plenty of other studies concerns the sorting of certain cell populations that need to be dis-tinguished carefully.To select and capture target cells from the rest is not an issue speci?c to single cell studies.However,it is the uttermost resolution offered by single cell sequencing that makes the isolation step extremely signi?cant,which,if taken improperly,would complicate and mislead the subsequent analyses.Single cell sequenc-ing of the circulating tumor cells (CTCs)is a case deserving special attention.The CTCs are so rare among
massive numbers of blood cells [5]that the accurate identi?cation of CTCs turns out to be the most important step in the isolation process.To ?nd an approach that can enrich CTCs with both low false positive and false negative rate becomes the prerequisite when monitoring and understanding tumor status by sequencing them.Another source of cells that need to be carefully isolated is cancer stem cells (CSCs).Lots of efforts have been spent on the methods to hunt CSCs from various tumor tissues [6,7].Whatever mechanism the method bases on,the CSCs enriched will suffer from a certain degree of non-stem-cell contamination,which happens to be the barrier that hinders the application of single cell sequencing in CSCs study [8].In a word,the accurate isolation of single target cells is a critical step proceeding single cell analyses.
So far various techniques have been developed to isolate single cells,such as micromanipulation,?ow cytometry,micro?uidic devices,laser capture microdissection,and optical tweezers [9].All of these techniques have strengths and weaknesses so that they are applied in different cir-cumstances (Table 1).
As a major form of micromanipulation,manual cell picking using micro capillary pipettes is the most widely used method to isolate single cells,not only because it needs no additional equipment,but also because this method is easy to apply to almost all circumstances.However,one can hardly use this method to isolate hun-dreds or thousands of single cells,which requires intensive labor work due to the low throughput feature.Besides,a standardized quality control is hard to achieve since the operator is susceptible to subjective judgments.
The optical tweezers,sometimes known as the single-beam gradient force trap,are also a form of micromanip-ulation [10].With a highly focused laser beam exerting attracting forces,the optical tweezers are able
to
Fig.1Single cell sequencing techniques
manipulate single cells in a contact-free way.However,the disadvantages of optical tweezers are the heating and photodamage effects brought by the laser beam,as well as the problem of low throughput.
To increase the throughput of micromanipulation,some laboratories automate the cell picking process by deposit-ing target cells in an array of micro-fabricated wells [11].After a serial of dilution,each well will host at most one cell.Following validation under a microscope,each target cell can be either pipetted into a new reaction tube or lysed in situ.Integrated with computerized imaging platform and robotic arms for micro-pipetting,the system can handle a large number of single cells within a short time period.Another strategy to isolate single target cells is by FACS [12].It is used most commonly to enrich target cells with speci?c surface markers or simply sort single cells from a suspension.The requirement for a relatively large number of cells as the starting material is a disadvantage for using FACS.Additionally,the strong vibrations used in FACS to break the stream into individual droplets may damage the cell,resulting in membrane leakage and loss of mRNA or even DNA.
Compared to other techniques,single cell sequencing combined with micro?uidic platforms shows a promising future [13–15].The advantages of isolation by micro-?uidics are as follows:(1)by a smart design of the system layout for ?ne control,it can isolate single cells in a parallel,high-throughput manner;(2)all steps are done in a closed system and it eliminates possible pipette errors,transfer loss,and contamination from the environment;(3)the system can easily incorporate the following DNA and RNA ampli?cation steps after isolation of single target cells;(4)more importantly,a small reaction vol-ume inherent to the micro?uidic chip improves the ampli?cation ?delity and reaction ef?ciency,which are key to single cell sequencing.Many laboratories have designed micro?uidic platforms to match their research objectives [16,17]and some of these platforms have been commercialized [18].With more efforts joining the rally to apply this prospective technology,we expect more automated systems emerging for single cell sequencing,which will free researchers from sample preparation steps.
Clearly different from the preceding methods,laser capture microdissection (LCM)can isolate single cells not in ?uid suspension [19].By cutting single cells in tissue sample sections,the spatial information of these target cells,otherwise would lose in cell suspension,can be retrieved.In this way,LCM is quite suitable for cancer studies.However,since cells in the tissue sample have close contact with their neighbors,single cell isolated by LCM may suffer from genetic material contaminations from the surrounding cells.
T a b l e 1S i n g l e c e l l i s o l a t i o n t e c h n i q u e s
T e c h n i q u e s
C e l l i d e n t i ?c a t i o n
I s o l a t i o n m e t h o d
A u t o m a t i o n F e a t u r e s
L i m i t a t i o n s M i c r o m a n i p u l a t i o n w i t h m i c r o c a p i l l a r i e s
M o r p h o l o g y /?u o r e s c e n c e
M o u t h p i p e t t e /m i c r o p i p e t t e
N o
G o o d o p e r a b i l i t y a n d h i g h c o n ?d e n c e
L o w t h r o u g h p u t ,s u s c e p t i b l e t o s u b j e c t i v e j u d g m e n t s
M i c r o m a n i p u l a t i o n w i t h m i c r o w e l l s
M o r p h o l o g y /?u o r e s c e n c e
M i c r o p i p e t t e
Y e s
H i g h -t h r o u g h p u t ,n a n o l i t e r v o l u m e
C r o s s c o n t a m i n a t i o n b e t w e e n w e l l s ,l o w e f ?c i e n c y c a u s e d b y l o w l o a d i n g d e n s i t y
O p t i c a l t w e e z e r
M o r p h o l o g y /?u o r e s c e n c e
O p t i c a l t r a p
N o
C o n t r o l l i n g c e l l s w i t h o u t c o n t a c t
L o w t h r o u g h p u t ,p h o t o d a m a g e o f c e l l s c a u s e d b y l a s e r b e a m
L a s e r c a p t u r e m i c r o d i s s e c t i o n
M o r p h o l o g y /?u o r e s c e n c e
L a s e r b e a m c u t t i n g
N o
I s o l a t i n g c e l l s i n t i s s u e s e c t i o n s
G e n e t i c m a t e r i a l c o n t a m i n a t i o n f r o m n e i g h b o r i n g c e l l s
F l u o r e s c e n c e -a c t i v a t e d c e l l s o r t i n g F l u o r e s c e n c e /s i z e
E l e c t r i c c h a r g e Y e s H i g h -t h r o u g h p u t ,s o r t i n g c e l l s w i t h s p e c i a l m a r k e r s L a r g e a m o u n t o f s t a r t i n g m a t e r i a l n e e d e d ,l e a k y c e l l c a u s e d b y s t r o n g v i b r a t i o n
M i c r o ?u i d i c s M o r p h o l o g y /?u o r e s c e n c e
F l u i d s c o n t r o l l e d b y m i c r o v a l v e
Y e s
H i g h -t h r o u g h p u t ,n a n o l i t e r v o l u m e ,i n t e g r a t e d w i t h a m p l i ?c a t i o n
P r o f e s s i o n a l s k i l l s n e e d e d t o b u i l d t h e s e t u p ,l i m i t e d c h o i c e s f o r n o n -e x p e r t s
2Single cell genome sequencing
One human diploid single cell contains46chromosomes, whose DNA weigh only*6pg,far too low from the amount of ng*l g DNA required for the NGS.Moreover, every gene of the genome in a single normal cell only has two copies(one copy in each haploid gamete cell).As a result,a precise,unbiased ampli?cation of the DNA is critical to genome sequencing of single cells.However, ampli?cation using traditional PCR suffers from severe biases and allelic dropout across the genome when it is applied to single cells.Lots of attempts were made,mostly modifying the PCR,such as linker-adapter PCR(LA-PCR)
[20],primer extension preampli?cation PCR(PEP-PCR)
[21],degenerate oligonucleotide-primed PCR(DOP-PCR)
[22],and its variant displacement DOP-PCR(D-DOP-PCR)[23]et al.,to improve the ef?ciency and?delity of whole genome ampli?cation(WGA).Various commercial WGA kits have been developed based on these methods and applied to single cell genomic studies(Table2).For instance,with the GenomePlex WGA4kit(DOP-PCR), Navin and colleagues sequenced single breast cancer cells dissected from solid tumor tissues at a low coverage to study the copy number variation at whole genome level and reconstructed the evolution lineage of tumor progression [12].
Dean et al.[24]invented the multiple displacement ampli?cation(MDA)method.With random hexamer primers and phi29DNA polymerase,it can amplify DNA in a30°C isothermal reaction.As the key to MDA,phi29 DNA polymerase can extend the primers with high?delity and strong processivity,which exhibits powerful strand displacement ability during the new strand synthesis (Fig.2).In this scenario,the DNA polymerase would jack up the preceding strand when it moves along the DNA template,thus in turn creating new docking sites for the free primers and enzymes.Therefore,DNA synthesis can be performed simultaneously on both the original template and the growing amplicons,initiating a fast and multiplex ampli?cation of the genomic DNA.When the ampli?cation is done,the genomic DNA from a single cell can be ampli?ed to106folds,with the?nal products of12kb on average,and the longest DNA fragment can reach100kb. The?rst application of MDA came in2005when researchers successfully ampli?ed and sequenced the gen-ome of a single Escherichia coli cell[25],which inspires great passion in microbiologists to sequence the genome of unculturable microorganisms.Recently,Mike McConnell and colleagues surveyed the genomes of110individual frontal cortex cells from three healthy human brains.Sur-prisingly,they found that a high proportion of neurons have megabase-scale de novo copy number variations[26].This study echoed the elevated level of aneuploidy found previously in both mouse and human neurons.Further-more,the mosaic CNV in human neurons is quite an evi-dence of tissue mosaicism,which needs to be investigated in other human tissues and can be an exciting research?eld for single cell genome sequencing.
However,MDA also suffers from strong biases and high allelic dropout rate across the genome.Additionally,the multiple displacement events make the reaction vulnerable to generating‘‘chimeras’’,resulting in much unwanted noise and false result when analyzing the sequencing data [27].
Zong et al.[28]reported a new ampli?cation method—multiple annealing and looping-based ampli?cation cycles (MALBACs)—that can amplify the genome of a single cell with high https://www.doczj.com/doc/b717978452.html,ing random primers?anked by a universal sequence,the full amplicons generated in the reaction seal themselves to form loops,protecting them from being ampli?ed again and ensuring that each new amplicon is replicated from the original templates(Fig.2). By performing?ve cycles of such linear ampli?cation before using the traditional PCR,the ampli?cation errors and biases can be greatly reduced,because the starting materials of the exponential ampli?cation are amplicon separately copied from the original template,which can even out the errors and biases introduced in an individual reaction.
With MALBAC,the researchers claimed they can achieve93%genome coverage(C19)for a single human cell at259sequencing depth,detecting not only digitized copy number variations(CNVs),but also single nucleotide variations(SNVs)at a low false positive rate.The new method was soon implemented in the single cell study of 99human sperms.They found that the meiotic recombi-nation events are non-uniformly distributed across the genome in the absence of selection pressure[29].
Later,Hou et al.[30]analyzed single human oocytes with MALBAC.In this study,by sequencing the?rst and second polar bodies and the oocyte pronuclei from the same female egg donors,the researchers can phase the genomes of these donors.Vice versa,the genome infor-mation of a female pronucleus of a fertilized egg can be deduced by sequencing the?rst and second polar bodies.In this pilot study,the researchers demonstrated the clinic value of genomic screening in in vitro fertilization(IVF). With single cell sequencing,it is possible to select healthy fertilized eggs for embryo transfer,which marks an important milestone in the application of single cell sequencing in translational medicine.
Meanwhile,Ni et al.[31]characterized the genome of single CTCs from lung cancer patients.They found CTCs from the same patient exhibit reproducible copy number variation patterns.What’s more,a certain degree of simi-larity is shared among the CNVs of CTCs from patients
with the same lung adenocarcinoma.The researchers fur-ther showed that SNVs on key oncogenes or tumor sup-pressor genes can be detected in single CTCs,paving the way for sequencing CTCs as a noninvasive method for cancer diagnosis and prognosis.
Again,MALBAC is still not perfect.The ultimate goal of single cell genome sequencing is to be as precise as bulk sequencing,offering accurate detection of CNVs,insertion and deletions (InDels),SNVs,and other structure varia-tions.Many laboratories are improving the single cell ampli?cation methods to achieve a higher ?delity and lower bias [18,32].With intensive efforts devoted into this area,we expect waves of technology advancements in single cell genome sequencing in the near future.
3Single cell transcriptome sequencing
As a powerful tool in genomic analysis,NGS also funda-mentally bene?ts transcriptomic studies [33–35].Tran-scriptome sequencing enables researchers to detect thousands of known and unknown transcripts in various kinds of tissues and cells [36,37],thus revealing differ-ential gene expression and diverse RNA splicing patterns.Like genomic sequencing,transcriptome sequencing by NGS also requires a large amount of RNA as the starting material.However,some basic biological processes involve only rare or even single cells,for example,the early embryonic development.Meanwhile,greater heter-ogeneity exists among individual cells at the transcrip-tomic level than at the genomic level,due to the fact that gene expression de?nes different cell types and it is a dynamic process that can be in?uenced by both spatial and temporal factors.To study the above problems necessitates single cell transcriptome sequencing.
To apply the NGS to single cell transcriptome analysis,mRNA from single cells must be reverse-transcribed to cDNA followed by cycles of PCR ampli?cation [38].The relief is that mRNA is not as rare as DNA in a single cell,and many of them exist in thousands of copies.How to perform reverse transcription (RT)and the subsequent conversion to double-strand DNA with high ef?ciency and low biases are key to successful single cell mRNA ampli?cation.
Actually,applying whole transcriptome pro?ling to single cells is a long-existing pursuit,while the ?rst attempts date back to the 1990s [39,40].One big advancement is the development of single cell microarray techniques [41,42].The disadvantage of microarray is that it cannot discover novel transcripts and alternative splicing isoforms.Besides,due to the low detection sensitivity,the microarray analysis would likely miss many key but low-level transcripts.
T a b l e 2S i n g l e c e l l a m p l i ?c a t i o n t e c h n i q u e s
M e t h o d s
S t e p s
R e a c t i o n t i m e (h )
P r o d u c t l e n g t h /m e d i u m (k b )
F e a t u r e s
L i m i t a t i o n s
C o m m e r c i a l k i t s
G e n o m e
M D A
2
*8–9
2–100/12
I s o t h e r m a l r e a c t i o n ,s t r a n d d i s p l a c e m e n t
A m p l i ?c a t i o n b i a s ,a l l e l e d r o p o u t ,‘‘c h i m e r a ’’s t r u c t u r e
Q i a g e n R E P L I -g ò
M A L B A C
3
*4
0.5–1.5/–
Q u a s i -l i n e a r a m p l i ?c a t i o n ,s t r a n d d i s p l a c e m e n t
A m p l i ?c a t i o n b i a s ,a l l e l e d r o p o u t
Y i k o n òg e n o m i c s
D O P -P C R
3
*4
0.1–1/0.4
D e g e n e r a t e o l i g o n u c l e o t i d e p r i m e r
L o w c o v e r a g e ,A m p l i ?c a t i o n b i a s ,a l l e l e d r o p o u t
S i g m a -A l d r i c h ò
G e n o m e P l e x ò
T r a n s c r i p t o m e
T a n g 2009
7
14
0.5–3/–
C o u n t i n g t r a n s c r i p t s b a s e d o n 30e n d o f m R N A
L o w c o v e r a g e o n 50e n d o f m R N A
N o n e
S m a r t -s e q /237
0.5–10/1.5–2D e t e c t i n g f u l l -l e n g t h m R N A w i t h s p e c i a l R T R e l a t i v e l y l o w s e n s i t i v i t y
I n c o r p o r a t e d b y F l u i d i g m ò
M e t h y l o m e
s c R R B S
6120.3–0.5/–
O n e t u b e r e a c t i o n
L o w c o v e r a g e o f C p G s i t e s
N o n e
Tang et al.[43]pioneered the?rst single-cell RNA sequencing(RNA-seq)technique based on previous methods for microarray analyses[41,42].In this work, they extended the reaction time of RT to obtain the?rst-strand cDNA as long as3kb.After the synthesis,a poly-A tail is added to the30end of the?rst-strand cDNA,which serves as a priming site for oligo-dT primer with another anchor sequence so that the second-strand cDNA can be synthesized.In the following steps,newly synthesized cDNA can be ampli?ed through PCR reactions based on the anchor https://www.doczj.com/doc/b717978452.html,pared to microarray analyses, this method expanded the spectrum of detected genes with high accuracy and effectively increased the proportion of full-length cDNA.This method was soon used to study many crucial biological processes.For example,they studied the transition of cells from blastocysts of inner cell mass to pluripotent embryonic stem cells and detected the key molecular events and changes in transcript variants,as well as the epigenetic modi?cations contributing to this switch[44].Recently,Yan et al.[45]measured the gene expression in124single cells from preimplantation embryos and single human embryonic stem cells.They successfully detected22,687expressed genes including 8,701long noncoding RNAs(lncRNAs)which was far more than the number detectable by cDNA microarray. Altogether,these?ndings provide important insights into the gene regulatory network governing human early embryonic development.
In2012,a novel single-cell RNA sequencing method, Smart-seq,was reported for its robust performance in
Isothermal
reaction
MALBAC MDA
of single cell WGA by MALBAC and MDA.Both methods begin with single cell lysis and DNA
DNA of a single cell to microgram level.MALBAC is composed of two rounds of ampli?cations: (blue)?anked by a universal sequence(orange)anneal to the templates.Extension of the primers generate the universal sequence on one end).The following5repeats of this cycle generate the full-amplicons sequence on both ends),whose3’ends are complementary to their5’ends.Then they seal themselves to ‘‘looping step’’in the end of each cycle.Such loop structure prevents the full-amplicons from being used full-amplicons are exponentially ampli?ed by traditional PCR cycles.MDA has one round of isothermal (blue).The phi29DNA polymerase enables the newly synthesized strand to displace the formerly synthesized primers will anneal to these displaced single-strands and continue such cycles of‘‘displace and anneal’’
detecting mRNA at full-length[46].This method is designed to improve the coverage of the50ends of mRNA [47].Ramsko¨ld and colleagues used a special reverse transcriptase derived from the Moloney murine leukemia virus(MMLV).Unlike other reverse transcriptases,this enzyme possesses two additional functions namely the terminal transferase activity and template switch ability [48].In Smart-seq,when the cDNAs are elongated to the50 end of mRNAs,MMLV can ligate several free cytosines to the end of cDNAs without templates owing to its terminal transferase activity.After this,the predesigned primers with guanine residues tailed by a sequence for the fol-lowing PCRs can anneal to the poly-C.Then MMLV switch templates and transcribe to the end of the oligonu-cleotide.Therefore,the newly synthesized cDNA suc-cessfully cover the50end of the mRNA,meanwhile it contains an anchor sequence for the second-strand syn-thesis.Through?nal PCR ampli?cation based on the pre-built priming sites and following sequencing libraries construction,the cDNAs are subject to high-throughput sequencing eventually.Smart-seq promotes coverage across mRNA transcripts,which enables detailed analyses of alternative transcript isoforms and identi?cation of sin-gle-nucleotide https://www.doczj.com/doc/b717978452.html,ing Smart-seq,Rickard Sandberg’s group studied the transcriptome of single melanoma CTCs.By comparison with individual primary melanocytes,they found289genes were remarkably up-regulated and436genes were down-regulated in CTCs. Meanwhile,with the transcriptome data,they identi?ed several upregulated transcripts related to proteins on cell membrane in CTCs,which may serve as promising bio-marker candidates for melanoma CTCs[46].Recently, they optimized the reaction and released the new version as Smart-seq2[49].It was reported that the new protocol dramatically promotes both the cDNA yielding and the detection sensitivity for rare transcripts,and it can reduce biases[50].With Smart-seq2,Deng et al.[51]explored which allele was selectively transcribed in single cells, unveiling a random and dynamic monoallelic expression pattern at the single-cell level.
Patel et al.[52]pro?led430cells from?ve primary glioblastomas by single cell RNA-seq and showed heter-ogeneous gene expression patterns within tumor tissue. They showed that transcriptome analysis by single cell RNA-seq can not only reveal differential gene expression patterns of tumor cells,but also infer large-scale CNVs to capture their genomic information.
One big improvement of single cell RNA-seq came with the endeavors combining single cell RNA-seq methods with micro?uidics.Aaron Streets and colleagues success-fully integrated Tang2009protocol in a micro?uidic sys-tem[17].The new system shows a better performance with increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based proto-cols.At the same time,Treutlein et al.[53]used a com-mercial micro?uidic single-cell RNA sequencing device (Fluidigm C1)[18]to sequence198single cells from dif-ferent stages of alveolar differentiation and reconstructed their lineage relationship.Shalek et al.[54]sequenced over 1,700primary mouse bone marrow-derived dendritic cells spanning several experimental conditions also with Flui-digm C1.Besides the substantial variation between iden-tically stimulated dendritic cells revealed by single cell RNA-seq analysis,they further found the so-called‘‘pre-cocicous’’cells,which responded to the stimulation earlier and then communicated the signal to others by paracrine.
Although single-cell RNA-seq techniques are gradually improving,there are still a few problems remained to be tackled in the future:(1)all of the methods mentioned above prefer to select mRNAs with poly-A tails and neglect other mRNAs such as histone mRNAs[55];(2)in most of these studies,the single cell were isolated from suspension,which always requires digestion and sometimes labeling of the target cells.Optimized protocols for sample preparation, which minimize the in?uence or harm to the cell state,are highly desirable;(3)it is dif?cult to apply the current methods to study target cells in their original context.The spatial information of cells is usually lost.One possible solution to overcome this dif?culty is to sequence cellular RNA in situ[56];Besides,Durruthy-Durruthy et al.[57] showed that the spatial information of single cells can be recovered by a clever strategy combining speci?c marker genes and three-dimensional principal component analysis. As a whole,technical advancements in these directions will certainly make big impacts for single cell transcriptome sequencing and its applications.
4Single cell methylome
Epigenetic modi?cation is a crucial determinant for gene expression because it greatly in?uences the DNA-binding af?nity of corresponding transcription factors(TFs)[58].As a major form of epigenetic modi?cation,DNA methylation is mediated by DNA cytosine methyltransferases,including Dnmt1,Dnmt3a,and Dnmt3b,which add methyls to the ?fth carbon atom on the cytosine ring forming5-methyl cytosine[59].DNA methylation plays a signi?cant role in genomic imprinting,X chromosome inactivation,and many other important biological processes[60,61].In genome, methylation often happens at the CpG sites.The clusters of CpG sites are called CpG islands(CGIs).Therefore,the methylation status of CGIs can effectively represent the genome-wide methylation landscape.
Reduced representation bisul?te sequencing(RRBS)was developed to analyze the methylation of CGIs at the genome-
wide level.In this method,the restriction enzyme MspI can recognize the CGIs and cut the genome into fragments at these sites,resulting in the enrichment of CGIs[62].Then these fragments are treated with bisul?te,which can convert the unmethylated cytosines to uracils while leave the methylated ones intact.After high-throughput sequencing and data ana-lysis,the information of DNA methylation can be retrieved [63].The RRBS technique has demonstrated its great advan-tages in sequencing DNA methylome of bulk cells and some studies have showed that it can be successfully applied to less than100cells[64,65].However,it was not until the recent invention of single cell RRBS(scRRBS)that researchers can analyze the methylome at single cell resolution.Guo and colleagues improved the conventional protocol of RRBS[63, 66]and managed to perform all reaction steps before bisul?te conversion in a single tube,which minimize the severe DNA loss in repeated puri?cation processes[67].With this method, researchers successfully detected in single cells an average 40%of mouse genome CGIs that can be detected by bulk RRBS.Recently,they pro?led the methylome of human early embryos in continuous stages[68].By analyzing the methyl-ation status of individual male and female pronuclei at the same time point after intra-cytoplasmic sperm injection with scRRBS,they found that the demethylation process is very heterogeneous at single-cell level.
However,scRRBS can only cover a small portion of CpG sites in the whole genome owing to the RRBS’s limitation. Therefore,a more ef?cient way to capture CpG sites will open a new window for the economic study of methylome and its adaptation to single cells is highly expected.What is more,how to alleviate dramatic DNA degradation during bisul?te conversion still remains unresolved.Innovative strategies are needed to overcome these problems and to improve the coverage of single cell methylome analyses. 5Future development and application
The breakthrough in single cell methylome sequencing sheds light on singe cell epigenetic analysis at the genome-wide level.Meanwhile,epigenetic events also include other modi?cations,such as histone methylation and acetylation. In addition,the epigenetic regulation involves cis-acting factors like enhancers and trans-acting factors like TFs, which can be measured by chromatin immunoprecipitation sequencing(ChIP-seq).To measure all these events in single cells is one major direction of future single cell analyses.
All the single cell sequencing techniques we discussed so far can depict only one dimension of the genetic infor-mation embodied in a single cell.None have been reported to be able to analyze multiple layers of information simultaneously.An integrated analysis of genomes,tran-scriptomes,and epigenomes will provide a comprehensive understanding of the complex interactions between differ-ent levels of the central dogma[69].For example,the transcriptome data of tumor re?ects how the expression pattern changes according to different variations in the genome.The combined analysis,if possible to perform on single cells,will for sure deliver new messages.
Another aspect that current single cell sequencing methods seldom consider is the spatial information.In situ sequencing can not only reveal the genome or transcriptome information, but also reserve the morphology and local environment of single cells[56,70].For instance,the‘‘niches’’where stem cells reside are thought to be the signal source that maintain stem cells in undifferentiated states and induce their self-renewal[71].Besides,spatial distribution of cells based on the three major body axes,dorsal/ventral,medial/lateral,and posterior/anterior conveys important information about tissue development[57].The ability of single cell sequencing with spatial information built-in will de?nitely reveal deeper insights into the cell-microenvironment interactions and the cell organization in complex tissues.
Single cell sequencing in micro?uidics have broad space for future developments.Establishing a standard protocol and robust system for single cell sequencing will make these techniques accessible to more research laboratories and ?elds.Thanks to the excellent plasticity,controllability with programmed mechanics inherent to micro?uidics,the appli-cation of them in single cell sequencing will prevail in basic research as well as clinical applications in the future.
To summarize,the next breakthrough in single cell sequencing lies in either the development of sequencing technology which requires less genetic materials,or better ampli?cation methods producing minimum errors and bia-ses.Great efforts are being made on both directions.Tech-nology innovation is still the driving force for the broad application of single cell sequencing.In the long future,we expect that the single cell techniques will become a powerful tool to unravel longstanding biomedical question. Acknowledgments This work was supported by the Recruitment Program of Global Youth Experts to Fan Bai.We thank Yanyi Huang, Fuchou Tang and Xiaoliang Sunney Xie from BIOPIC of Peking University for valuable discussions.
Con?ict of interest The authors declare that they have no con?ict of interest.
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CONTENTS[中文概要]
纳米生物效应与纳米毒理学中的剂量与表面化学效应关系
赵峰, 孟幻, 晏亮, 汪冰, 赵宇亮
生命过程是由一系列发生在纳米尺度上的程序化、多级次、多步骤的化学、物理或生物学过程组成. 有趣的是, 在构成细胞的亚细胞器中或它们之间发生的这些复杂过程, 很多需要对生物分子(如蛋白质、核酸等)进行纳米尺度空间上的精确调控, 以维持生命过程的正常进行. 因此, 理解在纳米尺度下物质与生命体系的相互作用, 对生命科学与纳米科学、化学、材料科学、医学、环境健康科学和毒理学等领域的交叉和融合, 将提供独特的视点和启迪. 本文从纳米化学的角度, 系统归纳影响纳米材料在体内的生物蓄积、作用器官(或靶器官)和体内毒性的关键因素, 主要集中在纳米表面化学修饰和剂量效应. 由于已有的纳米材料很多, 本文重点分析了碳纳米管、金属相关(金属和金属氧化物)纳米材料以及量子点在生物体内的蓄积规律、作用器官选择性及其体内毒理; 它们的剂量效应; 以及纳米表面化学修饰对其体内蓄积规律、作用器官选择性及其体内毒理的调控作用. 最后, 我们从纳米化学的角度讨论这个领域具有挑战性的科学问题以及建立概念性知识框架尚需要深入研究的方向. 这篇综述是我们将纳米毒理学领域的知识系统化的持续努力的一部分.
封面文章 p3
动态RNA m6A甲基化修饰及其调控mRNA剪接机制
杨莹, 孙宝发, 肖文, 杨鑫, 孙慧颖, 赵永良, 杨运桂
RNA 6-甲基腺嘌呤(m6A)动态修饰酶的研究及RNA m6A免疫沉淀及高通量测序技术(MeRIP-seq/m6A-seq)的快速发展, 为揭示m6A在调控RNA加工、个体发育、分化、代谢及生殖等生物学功能提供了新契机. 催化m6A修饰形成的甲基转移酶复合物至少包括了催化亚基METTL3和METTL14及调控亚基WTAP; 加双氧酶家族蛋白FTO和ALKBH5作为m6A去甲基化酶催化m6A去甲基化; 而m6A结合蛋白主要分布于细胞质的YTH结构域家族YTHDF1-3和细胞核的YTHDC1-2. 扰动上述调控m6A动态的修饰酶活性可导致上千基因的表达变化, 并影响mRNA稳定性及剪接加工等. 本文综述了近期m6A甲基转移酶、去甲基化酶和结合蛋白的重要研究进展, 并讨论了m6A调控RNA加工代谢尤其是pre-mRNA剪接的潜在作用机制.
评述 p21
单细胞测序: 技术, 应用和未来发展
薛瑞栋, 李若岩, 白凡
二代测序技术的快速发展正在持续不断地改变着基因组学、转录组学、表观基因组学等的研究. 值得注意的是, 单细胞操纵和扩增技术的进步使得二代测序技术能被很好地应用到单细胞水平的研究中. 单细胞基因组测序可以帮助人们在全基因组信息层面研究肿瘤进化, 精(卵)子形成过程以及体细胞镶嵌现象等; 单细胞转录组测序帮助人们揭示了早期胚胎发育、细胞分化、细胞重编程等过程中基因表达的动态变化情况; 单细胞表观遗传组测序技术刚刚起步, 在癌症和干细胞研究等领域都有很大的应用前景. 同时, 许多研究正在尝试发展其他的单细胞组学研究技术. 要使单细胞测序技术能有更广泛的应用, 未来的技术需要具备以下特点: (1) 高准确度和忠实度地对信息进行扩增; (2) 能对同一个单细胞进行多个组学性质的分析; (3) 高程度的自动化和标准化的操作流程. 这些方面的技术进步和优化能不断降低单细胞测序技术在普通实验室的应用门槛. 单细胞测序技术的广泛应用也将对未来的生物医学研究带来显著改变.
评述 p33
探讨核物理学中的人择思想
Ulf-G. Mei?ner
讨论了较轻的且和生命相关的元素, 例如碳元素和氧元素的产生对于与核物理学相关的标准模型中参数变化的敏感性. 手征有效场理论可以系统精确地描述两核、三核以及四核之间的作用力, 同时可以计算其随轻夸克质量和电磁精细结构常数的变化. 运用手征核有效场理论和蒙特卡罗模拟方法可以进一步计算原子核的性质, 特别是碳原子的Hoyle态, 该态在炽热远古星体产生与生命相关元素的过程中起了重要作用. 另外, 本文讨论了3α过程对自然界基本常数的依赖性, 以及关于宇宙的人择观点的一些启示.
评述 p43
过氧化氢在燃料电池中的应用与展望
安亮, 赵天寿, 闫晓晖, 周学龙, 谈鹏
发展新的能源技术是解决全球能源与环境问题的关键. 作为新一代能源转换技术, 燃料电池可将燃料中的化学能直接转换为电能, 它是一种高效、清洁的能源转换系统, 必将在未来得到广泛应用. 燃料电池需要氧气作为氧化剂, 为此在诸如太空、水下等无氧环境中, 电池系统需要携带高压氧气储存系统. 因此, 常压下液态过氧化氢已经被广泛地应用在燃料电池中代替气态氧作为氧化剂. 另外, 过氧化氢还可以在燃料电池中通过氧化反应释放电子, 从而可以作为燃料. 本文着重介绍了过氧化氢燃料电池的最新进展, 包括工作原理、系统设计及电池性能, 并展望了今后的研究方向.
评述 p55
肿瘤基因组学与国际肿瘤基因组协作联盟
胡学达, 杨焕明, 赫捷, 吕有勇
DNA序列的变异是所有肿瘤细胞发生的重要的分子层面的原因, 目前学术界已经有能力对一定规模的癌症队列样本开展全基因组变异图谱的分析. 国际肿瘤基因组协作联盟(ICGC)于2007年成立并启动了全球范围的肿瘤基因组研究工作. ICGC提出对50种癌症, 总计25000例患者样本绘制体细胞基因突