The Orphanage (2007 film)

The Orphanage (Spanish: El orfanato) is a 2007 gothic supernatural horror film and the debut feature of J. A. Bayona. The film stars Belén Rueda as Laura, Fernando Cayo as her husband, Carlos, and Roger Príncep as their adopted son Simón. The plot centers on Laura, who returns to her childhood home, an orphanage. Laura plans to turn the house into a home for disabled children, but after an argument with Simón, he goes missing.

The film's script was written by Sergio G. Sánchez in 1996 and brought to the attention of Bayona in 2004. Bayona asked his long-time friend, director Guillermo del Toro, to help produce the film and to double its budget and filming time. The Orphanage is an international co-production between Spain and Mexico. Bayona wanted the film to capture the feel of 1970s Spanish cinema; he cast Geraldine Chaplin and Belén Rueda, who were later praised for their roles in the film.

The film opened at the Cannes Film Festival on 20 May 2007, where it received a standing ovation lasting more than 10 minutes. It received domestic critical acclaim in Spain, and won seven Goya awards, including Original Screenplay and New Director. On its North American release, The Orphanage was praised by English-speaking critics, who described the film as well directed and well acted, and noted the film's lack of "cheap scares"; subsequently, New Line Cinema bought the rights to the film for an American remake.

## Plot

In Spain, a young girl named Laura García Rodríguez is adopted from an orphanage. 30 years later, adult Laura returns to the closed orphanage, accompanied by her husband, Carlos Sánchez Rivera, and their seven-year-old son, Simón. She plans to reopen the orphanage as a facility for disabled children. Simón claims to have befriended a boy named Tomás, and draws pictures of him as a child wearing a sack mask. Social worker Benigna Escobedo visits the house to inquire after Simón, and it is revealed that Laura and Carlos adopted Simón and that he is HIV positive. Incensed at Benigna's intrusion, Laura asks her to leave. Later that night, Laura finds Benigna in the orphanage's coal shed, but Benigna flees the scene. Later, Simón teaches Laura a game which grants its winner a wish. Clues lead the two to Simón's adoption file. Simón becomes angry, and says that his new friend told him that Laura is not his biological mother and that he is going to die soon.

During a party for the orphanage's opening, Laura and Simón argue, and Simón hides from her after she slaps him across the face in a fit of frustration, which she immediately regrets. While looking for him, she encounters a child wearing a sack mask who shoves her into a bathroom and locks her inside. When Laura escapes, she realizes that Simón is missing and is unable to find him. That night, Laura hears several loud crashes within the walls of the orphanage. Police psychologist Pilar suggests to Laura and Carlos that Benigna may have abducted Simón.

Six months later, Simón is still missing. While searching for him, Laura spots Benigna, who is then struck and killed by an ambulance. The police find evidence that Benigna worked at the orphanage, and that she had a son named Tomás, who also lived there but was kept hidden due to his facial deformity. A few weeks after Laura was adopted, the orphans stole the mask that Tomás wore to conceal his deformed face. Embarrassed, Tomás refused to leave his hiding place in a nearby sea cave, and the rising tide drowned him.

Laura asks for the assistance of a medium named Aurora in the search for Simón. Aurora conducts a seance during which she claims to see the ghosts of the orphans crying for help. Laura discovers the remains of the orphans she grew up with in the orphanage. Benigna poisoned their meals and killed them for having caused Tomás's death and hid their remains in the orphanage's coal shed. Unable to cope with the situation, Carlos leaves the orphanage.

Laura makes the orphanage look as it did thirty years ago and attempts to contact the children's spirits by playing one of their old games. The spirits lead her to the door of a hidden underground room. Inside is Simón's corpse, wearing Tomás's mask. Laura finally realizes what happened: while searching for Simón the night he disappeared, Laura moved pieces of construction scaffolding, blocking the entrance to the secret room. The crashes that night were caused by Simón trying to get out. He fell and fatally broke his neck.

Laura appears to take an overdose of sleeping pills. Then, apparently dying, she begs to be with Simón again and the children's spirits appear, with Simón among them. Simón tells Laura that his wish was for her to stay and take care of the orphans, she then happily tells them a story. Sometime later, Carlos visits a memorial to Laura, Simón and the orphans. Carlos returns to the orphans' old bedroom and finds a medallion that he had given to Laura. He turns to look as the door opens, and he smiles.

## Cast

- Belén Rueda as Laura García Rodríguez, the wife of Carlos and adoptive mother of Simón. Laura returns to the orphanage where she spent some of her youth to turn it into a home for disabled children. Mireia Renau portrays the younger Laura.
- Fernando Cayo as Carlos Sánchez Rivera, the husband of Laura and adoptive father of Simón.
- Roger Príncep as Simón Sánchez Rivera, the young adopted son of Laura and Carlos. Simón meets new imaginary friends in the orphanage and eventually threatens to run away with them.
- Mabel Rivera as Pilar, the head police psychologist who eventually discovers the truth of who Benigna really is.
- Montserrat Carulla as Benigna Escobedo, a former worker at the orphanage who identifies herself as a social worker. When she is seen young in the film, she is portrayed by Carol Suárez. Production companies working with Bayona tried to urge him to keep this character alive until the end of the film.
- Geraldine Chaplin as Aurora, the medium brought in to help find Simón when the police can't find him.
- Andrés Gertrúdix as Enrique, Aurora's sound technician.
- Edgar Vivar as Professor Leo Balabán, a parapsychology expert who puts Laura in contact with Aurora and directs the spirit session in her house.
- Óscar Casas as Tomás, the deformed son of Benigna who Simón claims to have befriended.

## Production

### Development

This was an international co-production film between Spain and Mexico. The first draft of the script of The Orphanage was written by Sergio G. Sánchez in 1996. Sánchez was not sure why he chose to write a genre film for the screenplay, as he explains, "I ended up writing a film in the style of those I liked as a kid, movies like Poltergeist, The Omen, and Rosemary's Baby which I ruined on the first VCR we owned at home." Sánchez revealed the literary influences underlying his writing of the script, such as The Turn of the Screw and Peter Pan. Sánchez originally wanted to direct the script but he was repeatedly turned down by various Spanish production companies. While Sánchez was working on the short film 7337 in 2004, he met with director Juan Antonio Bayona and offered him the script to direct. Bayona accepted the opportunity because he felt that a fantasy themed script like that of The Orphanage would allow him freedom as a director, saying the fantasy genre was a great tool for learning as it "allows manipulation of space and time as we wish or the use of certain camera moves with an immediate efficiency".

Bayona cut parts of the script, including the outcome of the other orphanage children, because he wanted to focus on the character of Laura and what happens to her. To create the film as he wanted, Bayona had to double both the film's budget and the amount of filming time. To accomplish this, Bayona received help from fellow film director Guillermo del Toro, whom he had met at Festival de Cine de Sitges when del Toro was presenting his film Cronos (1993). Del Toro offered to co-produce the film as soon as he learned about it. For the rest of his crew, Bayona worked with his regular team that he worked with on commercials and music videos.

### Casting

During casting discussions between Bayona and del Toro, Bayona expressed a desire to see Belén Rueda in the lead. Bayona admired her after seeing her performance as Julia in Alejandro Amenabar's The Sea Inside. Del Toro appreciated this choice, as he admired her as an actress and liked that Bayona was casting her against type. Bayona asked Rueda to watch The Innocents and Close Encounters of the Third Kind to prepare for the role. Another actress Bayona desired for the film was Geraldine Chaplin in the role of Aurora, the medium. Bayona stated that he wanted the film to have "the mood of 70s Spanish cinema and Geraldine starred in one of the best movies of that decade, Carlos Saura's The Secret of Anna, as the ghost of the mother. It made sense to have her play the medium." Bayona was nervous about filming with Chaplin. To break that tension, on the first day of shooting with her Bayona hid under a bed during a scene which required Chaplin to portray Aurora kneeling down near it in the dark, and he grabbed Chaplin's leg when she did so. Aurora's scream in the film is Chaplin's actual frightened scream as Bayona grabbed her. Roger Príncep's test-screening was one of the first for the role of Simón. Bayona test-screened over four-hundred children during two months before making his choice to cast Princep. Edgar Vivar was cast in role of Balabán; Bayona knew him through his work on the Mexican television series El Chavo and sent him an invitation for the role through e-mail.

### Filming

Production on The Orphanage began on May 15, 2006 in Llanes, Asturias. This location was chosen due to the area's diverse natural settings that include beaches, caves, cliffs, forests, a small village, and the Partarríu Manor where the orphanage scenes take place. The orphanage was an old colonial house from the end of the nineteenth century. Bayona wanted to use certain cinematographic techniques that were impossible to achieve in the house, so several parts of the house were reconstructed in sound stages. After four weeks in Llanes, the team moved to Barcelona to finish up the last ten weeks of filming in sound stages, making over 80% of the film there. Bayona showed the films La residencia and The Innocents to his director of photography on the film, to make special notice of the Scope lensing used in both films.

## Release

The Orphanage premiered at the Cannes Film Festival on May 20, 2007. The film was positively received with a ten-minute ovation from the audience. The film's Spanish debut took place at the Sitges Film Festival on October 4, 2007 where it opened the festival. The Orphanage premiered in Spain on September 10, 2007. The Orphanage was released in Spain on September 10, 2007 and was immensely successful in Spain after an \$8.3 million four-day launch from 350 screens. The film was the second highest-grossing debut ever for a Spanish film and was the biggest opening of the year, making it even larger than the worldwide success of the Spanish-Mexican film Pan's Labyrinth. It opened in limited release in the United States on December 28, 2007 and had a wide release on January 11, 2008. It opened in Mexico on January 25, 2008 and earned over \$11,000,000 at the box office.

In Spain, the film was nominated for 14 Goya Awards, including Best Picture and ended up winning awards for Best Art Direction, Best Director of Production, Best Makeup and Hair, Best New Director, Best Screenplay – Original, Best Sound Mixing, and Best Special Effects. The Orphanage was chosen by the Spanish Academy of Films as Spain's nominee for the 2007 Academy Award for Best Foreign Film, but ultimately did not end up as one of the five final nominees in that category. The Orphanage was picked up by Picturehouse at the Berlin Film Festival for American distribution.

In Iran, the film won Crystal Simorgh for Best Director at the "Seeking the Truth" section of the 27th Fajr International Film Festival.

### Home media

The Orphanage was released on DVD and Blu-ray on April 22, 2008 for a Region 1 release by New Line Cinema. Both discs featured the same bonus features. A Region 2 version was released on DVD by Optimum Releasing on July 21, 2008.

### Remake

In 2007, New Line Cinema bought the rights to produce an English-language remake with Guillermo del Toro as producer. On remakes, director Bayona noted that "The Americans have all the money in the world but can't do anything, while we can do whatever we want but don't have the money" and "The American industry doesn't take chances, that's why they make remakes of movies that were already big hits". On August 4, 2009, Larry Fessenden was announced as the director of the American remake. Fessenden later announced that he would not be involved with directing the remake, stating "Working on the script with Guillermo was a very exciting experience, but then I got into a casting miasma and that's where the thing is; I think they're gonna do it another way, actually. So I think I'm out of it. Hopefully they'll still use my script, but I'm not sure I'm directing it anymore". In January 2010, Mark Pellington replaced Larry Fessenden as director of the project.

On August 5, 2011, Guillermo del Toro stated that the remake would reflect his original vision for the film, and that it had been planned even when the first version was in production. "Even when we produced the Spanish movie, I had intended to remake it because we had a very different screenplay that, because of money and time, got turned into the movie you saw – which is great, but there was this other structure for the original script that I wanted to try. So even before we shot the first film it was an economic decision, a pre-existing creative decision, to change it." Del Toro also praised the new film's director. "We have Mark Pellington attached as director – I'm a big fan of his The Mothman Prophecies and his video work – and we are out to actors, so we're hoping to get things going soon." On August 30, 2011, it was reported that American actress Amy Adams was in talks to star as Laura, the main character, who was played by Belén Rueda in the original film. It was also stated that the current incarnation of the remake screenplay had been written by Larry Fessenden and Sergio G. Sánchez, the sole writer of the original film.

## Reception

### Critical response

The Orphanage was received very well by American critics on its original release. The film has an 87% approval rating on Rotten Tomatoes based on 178 reviews, with an average rating of 7.4/10. The critical consensus reads, "Deeply unnerving and surprisingly poignant, The Orphanage is an atmospheric, beautifully crafted haunted house horror film that earns scares with a minimum of blood." At Metacritic, the film has received an average score of 74 out of 100, based on 33 reviews from mainstream critics, indicating "generally favorable reviews".

Critics praised the film for its lack of cheap scares. Film critic for the Chicago Sun Times Roger Ebert approved of the film claiming it to be "deliberately aimed at viewers with developed attention spans. It lingers to create atmosphere, a sense of place, a sympathy with the characters, instead of rushing into cheap thrills". Bill Goodykoontz of the Arizona Republic echoed this statement noting, "Bayona never lets The Orphanage descend into cheap horror. The scares here are expertly done and, placed in the context of Laura's state of mind, well-earned, perhaps even explainable (or not)." Peter Howell of the Toronto Star wrote, "The year's best horror picture is also one of the simplest. The Orphanage makes little if any use of digital tricks to present its numerous terrors." The casting of Chaplin and Rueda was praised while the role of Carlos was called dull. Goodykoontz noted the role of Carlos, saying "Cayo is rather pedestrian as Carlos, but he isn't given that much to do". Michael Phillips of the Chicago Tribune shared this opinion saying, "Rueda has a great pair of peepers for this assignment. When she looks one way and then the other, while skulking through the hallways of her childhood home, every nerve-wracking whatwasthat? registers, and how. Cayo is pretty dull by comparison, but Chaplin certainly is not." Liam Lacey of The Globe and Mail praised Rueda stating, "The strongest appeal of the film is the brooding, intense performance by Spanish actress, Belen Rueda."

A negative review came from Lacey of The Globe and Mail, who felt that at "[the film's] core, it seems intended as a sympathetic drama of a bereaved mother, who may have slipped into madness. What's even more disquieting is the persistent undercurrent of exploitation – the mixture of grief and jarring shock effects and the pitiless use of a disfigured child as a source of horror." A.O. Scott of The New York Times claimed the film to be a "diverting, overwrought ghost story" and that it "relies on basic and durable horror movie techniques". Jack Matthews of New York Daily News found the ending of the film to be one of the worst of the season, but praised the acting of Belén Rueda.

The Orphanage listed as one of the top 10 best films of 2007 by several critics, including Lawrence Toppman of the Charlotte Observer, Marc Doyle of Metacritic and Tasha Robinson of The A.V. Club. Anthony Lane of The New Yorker included the film on his top ten list of 2008. In the early 2010s, Time Out conducted a poll with several authors, directors, actors and critics who have worked within the horror genre to vote for their top horror films. The Orphanage placed at number 76 on their top 100 list.

### Accolades

\|- \| rowspan = "21" align = "center" \| 2008 \|\| rowspan = "14" \| 22nd Goya Awards \|\| colspan = "2" \| Best Film \|\| \|\| rowspan = "14" \| \|- \| Best Actress \|\| Belén Rueda \|\| \|- \| Best Supporting Actress \|\| Geraldine Chaplin \|\| \|- \| Best Original Screenplay \|\| Sergio G. Sánchez \|\| \|- \| Best New Actor \|\| Roger Príncep \|\| \|- \| Best New Director \|\| J. A. Bayona \|\| \|- \| Best Editing \|\| Elena Ruiz \|\| \|- \| Best Art Direction \|\| Josep Rosell \|\| \|- \| Best Production Supervision \|\| Sandra Hermida \|\| \|- \| Best Sound \|\| Xavi Mas, Marc Orts, Oriol Tarragó \|\| \|- \| Best Special Effects \|\| David Martí, Montse Ribé, Pau Costa, Enric Masip, Lluís Castells, Jordi San Agustín \|\| \|- \| Best Costume Design \|\| María Reyes \|\| \|- \| Best Makeup and Hairstyles \|\| Lola López, Itziar Arrieta \|\| \|- \| Best Original Score \|\| Fernando Velázquez \|\| \|- \| rowspan = "2" \| 17th Actors and Actresses Union Awards \|\| Best Film Actress in a Leading Role \|\| Belén Rueda \|\| \|\| rowspan = "2" \| \|- \| Best Film Actress in a Minor Role \|\| Geraldine Chaplin \|\| \|- \| 13rd Forqué Awards \|\| colspan = "2" \| Best Film \|\| \|\| align = "center" \| \|- \| rowspan = "4" \| 21st European Film Awards \|\| colspan = "2" \| Best European Film \|\| \|\| rowspan = "4" \| \|- \| Best European Actress \|\| Belén Rueda \|\| \|- \| Best European Cinematographer \|\| Óscar Faura \|\| \|- \| Best European Composer \|\| Fernando Velázquez \|\| \|- \| align = "center" \| 2009 \|\| 57th Silver Condor Awards \|\| colspan = "2" \| Best Ibero-American Film \|\| \|\| align = "center" \|

## Soundtrack

In 2007, the film score was composed by Fernando Velázquez and released on compact disc by Rhino Records in Spain. The score for the film was nominated for film awards including the Goya Award for Best Score. The soundtrack has not been released locally in North America or the United Kingdom and is only available by import.

### Track listing

## See also

- List of ghost films
- List of horror films of 2007
- List of Spanish films of 2007
- List of submissions to the 80th Academy Awards for Best Foreign Language Film
- List of Spanish submissions for the Academy Award for Best Foreign Language Film

2007 supernatural horror film


Transcriptomics technologies

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated.

The first attempts to study whole transcriptomes began in the early 1990s. Subsequent technological advances since the late 1990s have repeatedly transformed the field and made transcriptomics a widespread discipline in biological sciences. There are two key contemporary techniques in the field: microarrays, which quantify a set of predetermined sequences, and RNA-Seq, which uses high-throughput sequencing to record all transcripts. As the technology improved, the volume of data produced by each transcriptome experiment increased. As a result, data analysis methods have steadily been adapted to more accurately and efficiently analyse increasingly large volumes of data. Transcriptome databases getting bigger and more useful as transcriptomes continue to be collected and shared by researchers. It would be almost impossible to interpret the information contained in a transcriptome without the knowledge of previous experiments.

Measuring the expression of an organism's genes in different tissues or conditions, or at different times, gives information on how genes are regulated and reveals details of an organism's biology. It can also be used to infer the functions of previously unannotated genes. Transcriptome analysis has enabled the study of how gene expression changes in different organisms and has been instrumental in the understanding of human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted assays.

## History

Transcriptomics has been characterised by the development of new techniques which have redefined what is possible every decade or so and rendered previous technologies obsolete. The first attempt at capturing a partial human transcriptome was published in 1991 and reported 609 mRNA sequences from the human brain. In 2008, two human transcriptomes, composed of millions of transcript-derived sequences covering 16,000 genes, were published, and by 2015 transcriptomes had been published for hundreds of individuals. Transcriptomes of different disease states, tissues, or even single cells are now routinely generated. This explosion in transcriptomics has been driven by the rapid development of new technologies with improved sensitivity and economy.

### Before transcriptomics

Studies of individual transcripts were being performed several decades before any transcriptomics approaches were available. Libraries of silkmoth mRNA transcripts were collected and converted to complementary DNA (cDNA) for storage using reverse transcriptase in the late 1970s. In the 1980s, low-throughput sequencing using the Sanger method was used to sequence random transcripts, producing expressed sequence tags (ESTs). The Sanger method of sequencing was predominant until the advent of high-throughput methods such as sequencing by synthesis (Solexa/Illumina). ESTs came to prominence during the 1990s as an efficient method to determine the gene content of an organism without sequencing the entire genome. Amounts of individual transcripts were quantified using Northern blotting, nylon membrane arrays, and later reverse transcriptase quantitative PCR (RT-qPCR) methods, but these methods are laborious and can only capture a tiny subsection of a transcriptome. Consequently, the manner in which a transcriptome as a whole is expressed and regulated remained unknown until higher-throughput techniques were developed.

### Early attempts

The word "transcriptome" was first used in the 1990s. In 1995, one of the earliest sequencing-based transcriptomic methods was developed, serial analysis of gene expression (SAGE), which worked by Sanger sequencing of concatenated random transcript fragments. Transcripts were quantified by matching the fragments to known genes. A variant of SAGE using high-throughput sequencing techniques, called digital gene expression analysis, was also briefly used. However, these methods were largely overtaken by high throughput sequencing of entire transcripts, which provided additional information on transcript structure such as splice variants.

### Development of contemporary techniques

The dominant contemporary techniques, microarrays and RNA-Seq, were developed in the mid-1990s and 2000s. Microarrays that measure the abundances of a defined set of transcripts via their hybridisation to an array of complementary probes were first published in 1995. Microarray technology allowed the assay of thousands of transcripts simultaneously and at a greatly reduced cost per gene and labour saving. Both spotted oligonucleotide arrays and Affymetrix high-density arrays were the method of choice for transcriptional profiling until the late 2000s. Over this period, a range of microarrays were produced to cover known genes in model or economically important organisms. Advances in design and manufacture of arrays improved the specificity of probes and allowed more genes to be tested on a single array. Advances in fluorescence detection increased the sensitivity and measurement accuracy for low abundance transcripts.

RNA-Seq is accomplished by reverse transcribing RNA in vitro and sequencing the resulting cDNAs. Transcript abundance is derived from the number of counts from each transcript. The technique has therefore been heavily influenced by the development of high-throughput sequencing technologies. Massively parallel signature sequencing (MPSS) was an early example based on generating 16–20 bp sequences via a complex series of hybridisations, and was used in 2004 to validate the expression of ten thousand genes in Arabidopsis thaliana. The earliest RNA-Seq work was published in 2006 with one hundred thousand transcripts sequenced using 454 technology. This was sufficient coverage to quantify relative transcript abundance. RNA-Seq began to increase in popularity after 2008 when new Solexa/Illumina technologies allowed one billion transcript sequences to be recorded. This yield now allows for the quantification and comparison of human transcriptomes.

## Data gathering

Generating data on RNA transcripts can be achieved via either of two main principles: sequencing of individual transcripts (ESTs, or RNA-Seq) or hybridisation of transcripts to an ordered array of nucleotide probes (microarrays).

### Isolation of RNA

All transcriptomic methods require RNA to first be isolated from the experimental organism before transcripts can be recorded. Although biological systems are incredibly diverse, RNA extraction techniques are broadly similar and involve mechanical disruption of cells or tissues, disruption of RNase with chaotropic salts, disruption of macromolecules and nucleotide complexes, separation of RNA from undesired biomolecules including DNA, and concentration of the RNA via precipitation from solution or elution from a solid matrix. Isolated RNA may additionally be treated with DNase to digest any traces of DNA. It is necessary to enrich messenger RNA as total RNA extracts are typically 98% ribosomal RNA. Enrichment for transcripts can be performed by poly-A affinity methods or by depletion of ribosomal RNA using sequence-specific probes. Degraded RNA may affect downstream results; for example, mRNA enrichment from degraded samples will result in the depletion of 5’ mRNA ends and an uneven signal across the length of a transcript. Snap-freezing of tissue prior to RNA isolation is typical, and care is taken to reduce exposure to RNase enzymes once isolation is complete.

### Expressed sequence tags

An expressed sequence tag (EST) is a short nucleotide sequence generated from a single RNA transcript. RNA is first copied as complementary DNA (cDNA) by a reverse transcriptase enzyme before the resultant cDNA is sequenced. Because ESTs can be collected without prior knowledge of the organism from which they come, they can be made from mixtures of organisms or environmental samples. Although higher-throughput methods are now used, EST libraries commonly provided sequence information for early microarray designs; for example, a barley microarray was designed from 350,000 previously sequenced ESTs.

### Serial and cap analysis of gene expression (SAGE/CAGE)

Serial analysis of gene expression (SAGE) was a development of EST methodology to increase the throughput of the tags generated and allow some quantitation of transcript abundance. cDNA is generated from the RNA but is then digested into 11 bp "tag" fragments using restriction enzymes that cut DNA at a specific sequence, and 11 base pairs along from that sequence. These cDNA tags are then joined head-to-tail into long strands (\>500 bp) and sequenced using low-throughput, but long read-length methods such as Sanger sequencing. The sequences are then divided back into their original 11 bp tags using computer software in a process called deconvolution. If a high-quality reference genome is available, these tags may be matched to their corresponding gene in the genome. If a reference genome is unavailable, the tags can be directly used as diagnostic markers if found to be differentially expressed in a disease state.

The cap analysis gene expression (CAGE) method is a variant of SAGE that sequences tags from the 5’ end of an mRNA transcript only. Therefore, the transcriptional start site of genes can be identified when the tags are aligned to a reference genome. Identifying gene start sites is of use for promoter analysis and for the cloning of full-length cDNAs.

SAGE and CAGE methods produce information on more genes than was possible when sequencing single ESTs, but sample preparation and data analysis are typically more labour-intensive.

### Microarrays

#### Principles and advances

Microarrays usually consist of a grid of short nucleotide oligomers, known as "probes", typically arranged on a glass slide. Transcript abundance is determined by hybridisation of fluorescently labelled transcripts to these probes. The fluorescence intensity at each probe location on the array indicates the transcript abundance for that probe sequence. Groups of probes designed to measure the same transcript (i.e., hybridizing a specific transcript in different positions) are usually referred to as "probesets".

Microarrays require some genomic knowledge from the organism of interest, for example, in the form of an annotated genome sequence, or a library of ESTs that can be used to generate the probes for the array.

#### Methods

Microarrays for transcriptomics typically fall into one of two broad categories: low-density spotted arrays or high-density short probe arrays. Transcript abundance is inferred from the intensity of fluorescence derived from fluorophore-tagged transcripts that bind to the array.

Spotted low-density arrays typically feature picolitre drops of a range of purified cDNAs arrayed on the surface of a glass slide. These probes are longer than those of high-density arrays and cannot identify alternative splicing events. Spotted arrays use two different fluorophores to label the test and control samples, and the ratio of fluorescence is used to calculate a relative measure of abundance. High-density arrays use a single fluorescent label, and each sample is hybridised and detected individually. High-density arrays were popularised by the Affymetrix GeneChip array, where each transcript is quantified by several short 25-mer probes that together assay one gene.

NimbleGen arrays were a high-density array produced by a maskless-photochemistry method, which permitted flexible manufacture of arrays in small or large numbers. These arrays had 100,000s of 45 to 85-mer probes and were hybridised with a one-colour labelled sample for expression analysis. Some designs incorporated up to 12 independent arrays per slide.

### RNA-Seq

#### Principles and advances

RNA-Seq refers to the combination of a high-throughput sequencing methodology with computational methods to capture and quantify transcripts present in an RNA extract. The nucleotide sequences generated are typically around 100 bp in length, but can range from 30 bp to over 10,000 bp depending on the sequencing method used. RNA-Seq leverages deep sampling of the transcriptome with many short fragments from a transcriptome to allow computational reconstruction of the original RNA transcript by aligning reads to a reference genome or to each other (de novo assembly). Both low-abundance and high-abundance RNAs can be quantified in an RNA-Seq experiment (dynamic range of 5 orders of magnitude)—a key advantage over microarray transcriptomes. In addition, input RNA amounts are much lower for RNA-Seq (nanogram quantity) compared to microarrays (microgram quantity), which allows finer examination of cellular structures down to the single-cell level when combined with linear amplification of cDNA. Theoretically, there is no upper limit of quantification in RNA-Seq, and background noise is very low for 100 bp reads in non-repetitive regions.

RNA-Seq may be used to identify genes within a genome, or identify which genes are active at a particular point in time, and read counts can be used to accurately model the relative gene expression level. RNA-Seq methodology has constantly improved, primarily through the development of DNA sequencing technologies to increase throughput, accuracy, and read length. Since the first descriptions in 2006 and 2008, RNA-Seq has been rapidly adopted and overtook microarrays as the dominant transcriptomics technique in 2015.

The quest for transcriptome data at the level of individual cells has driven advances in RNA-Seq library preparation methods, resulting in dramatic advances in sensitivity. Single-cell transcriptomes are now well described and have even been extended to in situ RNA-Seq where transcriptomes of individual cells are directly interrogated in fixed tissues.

#### Methods

RNA-Seq was established in concert with the rapid development of a range of high-throughput DNA sequencing technologies. However, before the extracted RNA transcripts are sequenced, several key processing steps are performed. Methods differ in the use of transcript enrichment, fragmentation, amplification, single or paired-end sequencing, and whether to preserve strand information.

The sensitivity of an RNA-Seq experiment can be increased by enriching classes of RNA that are of interest and depleting known abundant RNAs. The mRNA molecules can be separated using oligonucleotides probes which bind their poly-A tails. Alternatively, ribo-depletion can be used to specifically remove abundant but uninformative ribosomal RNAs (rRNAs) by hybridisation to probes tailored to the taxon's specific rRNA sequences (e.g. mammal rRNA, plant rRNA). However, ribo-depletion can also introduce some bias via non-specific depletion of off-target transcripts. Small RNAs, such as micro RNAs, can be purified based on their size by gel electrophoresis and extraction.

Since mRNAs are longer than the read-lengths of typical high-throughput sequencing methods, transcripts are usually fragmented prior to sequencing. The fragmentation method is a key aspect of sequencing library construction. Fragmentation may be achieved by chemical hydrolysis, nebulisation, sonication, or reverse transcription with chain-terminating nucleotides. Alternatively, fragmentation and cDNA tagging may be done simultaneously by using transposase enzymes.

During preparation for sequencing, cDNA copies of transcripts may be amplified by PCR to enrich for fragments that contain the expected 5’ and 3’ adapter sequences. Amplification is also used to allow sequencing of very low input amounts of RNA, down to as little as 50 pg in extreme applications. Spike-in controls of known RNAs can be used for quality control assessment to check library preparation and sequencing, in terms of GC-content, fragment length, as well as the bias due to fragment position within a transcript. Unique molecular identifiers (UMIs) are short random sequences that are used to individually tag sequence fragments during library preparation so that every tagged fragment is unique. UMIs provide an absolute scale for quantification, the opportunity to correct for subsequent amplification bias introduced during library construction, and accurately estimate the initial sample size. UMIs are particularly well-suited to single-cell RNA-Seq transcriptomics, where the amount of input RNA is restricted and extended amplification of the sample is required.

Once the transcript molecules have been prepared they can be sequenced in just one direction (single-end) or both directions (paired-end). A single-end sequence is usually quicker to produce, cheaper than paired-end sequencing and sufficient for quantification of gene expression levels. Paired-end sequencing produces more robust alignments/assemblies, which is beneficial for gene annotation and transcript isoform discovery. Strand-specific RNA-Seq methods preserve the strand information of a sequenced transcript. Without strand information, reads can be aligned to a gene locus but do not inform in which direction the gene is transcribed. Stranded-RNA-Seq is useful for deciphering transcription for genes that overlap in different directions and to make more robust gene predictions in non-model organisms.

Legend: NCBI SRA – National center for biotechnology information sequence read archive.

Currently RNA-Seq relies on copying RNA molecules into cDNA molecules prior to sequencing; therefore, the subsequent platforms are the same for transcriptomic and genomic data. Consequently, the development of DNA sequencing technologies has been a defining feature of RNA-Seq. Direct sequencing of RNA using nanopore sequencing represents a current state-of-the-art RNA-Seq technique. Nanopore sequencing of RNA can detect modified bases that would be otherwise masked when sequencing cDNA and also eliminates amplification steps that can otherwise introduce bias.

The sensitivity and accuracy of an RNA-Seq experiment are dependent on the number of reads obtained from each sample. A large number of reads are needed to ensure sufficient coverage of the transcriptome, enabling detection of low abundance transcripts. Experimental design is further complicated by sequencing technologies with a limited output range, the variable efficiency of sequence creation, and variable sequence quality. Added to those considerations is that every species has a different number of genes and therefore requires a tailored sequence yield for an effective transcriptome. Early studies determined suitable thresholds empirically, but as the technology matured suitable coverage was predicted computationally by transcriptome saturation. Somewhat counter-intuitively, the most effective way to improve detection of differential expression in low expression genes is to add more biological replicates rather than adding more reads. The current benchmarks recommended by the Encyclopedia of DNA Elements (ENCODE) Project are for 70-fold exome coverage for standard RNA-Seq and up to 500-fold exome coverage to detect rare transcripts and isoforms.

## Data analysis

Transcriptomics methods are highly parallel and require significant computation to produce meaningful data for both microarray and RNA-Seq experiments. Microarray data is recorded as high-resolution images, requiring feature detection and spectral analysis. Microarray raw image files are each about 750 MB in size, while the processed intensities are around 60 MB in size. Multiple short probes matching a single transcript can reveal details about the intron-exon structure, requiring statistical models to determine the authenticity of the resulting signal. RNA-Seq studies produce billions of short DNA sequences, which must be aligned to reference genomes composed of millions to billions of base pairs. De novo assembly of reads within a dataset requires the construction of highly complex sequence graphs. RNA-Seq operations are highly repetitious and benefit from parallelised computation but modern algorithms mean consumer computing hardware is sufficient for simple transcriptomics experiments that do not require de novo assembly of reads. A human transcriptome could be accurately captured using RNA-Seq with 30 million 100 bp sequences per sample. This example would require approximately 1.8 gigabytes of disk space per sample when stored in a compressed fastq format. Processed count data for each gene would be much smaller, equivalent to processed microarray intensities. Sequence data may be stored in public repositories, such as the Sequence Read Archive (SRA). RNA-Seq datasets can be uploaded via the Gene Expression Omnibus.

### Image processing

Microarray image processing must correctly identify the regular grid of features within an image and independently quantify the fluorescence intensity for each feature. Image artefacts must be additionally identified and removed from the overall analysis. Fluorescence intensities directly indicate the abundance of each sequence, since the sequence of each probe on the array is already known.

The first steps of RNA-seq also include similar image processing; however, conversion of images to sequence data is typically handled automatically by the instrument software. The Illumina sequencing-by-synthesis method results in an array of clusters distributed over the surface of a flow cell. The flow cell is imaged up to four times during each sequencing cycle, with tens to hundreds of cycles in total. Flow cell clusters are analogous to microarray spots and must be correctly identified during the early stages of the sequencing process. In Roche’s pyrosequencing method, the intensity of emitted light determines the number of consecutive nucleotides in a homopolymer repeat. There are many variants on these methods, each with a different error profile for the resulting data.

### RNA-Seq data analysis

RNA-Seq experiments generate a large volume of raw sequence reads which have to be processed to yield useful information. Data analysis usually requires a combination of bioinformatics software tools (see also List of RNA-Seq bioinformatics tools) that vary according to the experimental design and goals. The process can be broken down into four stages: quality control, alignment, quantification, and differential expression. Most popular RNA-Seq programs are run from a command-line interface, either in a Unix environment or within the R/Bioconductor statistical environment.

#### Quality control

Sequence reads are not perfect, so the accuracy of each base in the sequence needs to be estimated for downstream analyses. Raw data is examined to ensure: quality scores for base calls are high, the GC content matches the expected distribution, short sequence motifs (k-mers) are not over-represented, and the read duplication rate is acceptably low. Several software options exist for sequence quality analysis, including FastQC and FaQCs. Abnormalities may be removed (trimming) or tagged for special treatment during later processes.

#### Alignment

In order to link sequence read abundance to the expression of a particular gene, transcript sequences are aligned to a reference genome or de novo aligned to one another if no reference is available. The key challenges for alignment software include sufficient speed to permit billions of short sequences to be aligned in a meaningful timeframe, flexibility to recognise and deal with intron splicing of eukaryotic mRNA, and correct assignment of reads that map to multiple locations. Software advances have greatly addressed these issues, and increases in sequencing read length reduce the chance of ambiguous read alignments. A list of currently available high-throughput sequence aligners is maintained by the EBI.

Alignment of primary transcript mRNA sequences derived from eukaryotes to a reference genome requires specialised handling of intron sequences, which are absent from mature mRNA. Short read aligners perform an additional round of alignments specifically designed to identify splice junctions, informed by canonical splice site sequences and known intron splice site information. Identification of intron splice junctions prevents reads from being misaligned across splice junctions or erroneously discarded, allowing more reads to be aligned to the reference genome and improving the accuracy of gene expression estimates. Since gene regulation may occur at the mRNA isoform level, splice-aware alignments also permit detection of isoform abundance changes that would otherwise be lost in a bulked analysis.

De novo assembly can be used to align reads to one another to construct full-length transcript sequences without use of a reference genome. Challenges particular to de novo assembly include larger computational requirements compared to a reference-based transcriptome, additional validation of gene variants or fragments, and additional annotation of assembled transcripts. The first metrics used to describe transcriptome assemblies, such as N50, have been shown to be misleading and improved evaluation methods are now available. Annotation-based metrics are better assessments of assembly completeness, such as contig reciprocal best hit count. Once assembled de novo, the assembly can be used as a reference for subsequent sequence alignment methods and quantitative gene expression analysis.

Legend: RAM – random access memory; MPI – message passing interface; EST – expressed sequence tag.

#### Quantification

Quantification of sequence alignments may be performed at the gene, exon, or transcript level. Typical outputs include a table of read counts for each feature supplied to the software; for example, for genes in a general feature format file. Gene and exon read counts may be calculated quite easily using HTSeq, for example. Quantitation at the transcript level is more complicated and requires probabilistic methods to estimate transcript isoform abundance from short read information; for example, using cufflinks software. Reads that align equally well to multiple locations must be identified and either removed, aligned to one of the possible locations, or aligned to the most probable location.

Some quantification methods can circumvent the need for an exact alignment of a read to a reference sequence altogether. The kallisto software method combines pseudoalignment and quantification into a single step that runs 2 orders of magnitude faster than contemporary methods such as those used by tophat/cufflinks software, with less computational burden.

#### Differential expression

Once quantitative counts of each transcript are available, differential gene expression is measured by normalising, modelling, and statistically analysing the data. Most tools will read a table of genes and read counts as their input, but some programs, such as cuffdiff, will accept binary alignment map format read alignments as input. The final outputs of these analyses are gene lists with associated pair-wise tests for differential expression between treatments and the probability estimates of those differences.

Legend: mRNA - messenger RNA.

### Validation

Transcriptomic analyses may be validated using an independent technique, for example, quantitative PCR (qPCR), which is recognisable and statistically assessable. Gene expression is measured against defined standards both for the gene of interest and control genes. The measurement by qPCR is similar to that obtained by RNA-Seq wherein a value can be calculated for the concentration of a target region in a given sample. qPCR is, however, restricted to amplicons smaller than 300 bp, usually toward the 3’ end of the coding region, avoiding the 3’UTR. If validation of transcript isoforms is required, an inspection of RNA-Seq read alignments should indicate where qPCR primers might be placed for maximum discrimination. The measurement of multiple control genes along with the genes of interest produces a stable reference within a biological context. qPCR validation of RNA-Seq data has generally shown that different RNA-Seq methods are highly correlated.

Functional validation of key genes is an important consideration for post transcriptome planning. Observed gene expression patterns may be functionally linked to a phenotype by an independent knock-down/rescue study in the organism of interest.

## Applications

### Diagnostics and disease profiling

Transcriptomic strategies have seen broad application across diverse areas of biomedical research, including disease diagnosis and profiling. RNA-Seq approaches have allowed for the large-scale identification of transcriptional start sites, uncovered alternative promoter usage, and novel splicing alterations. These regulatory elements are important in human disease and, therefore, defining such variants is crucial to the interpretation of disease-association studies. RNA-Seq can also identify disease-associated single nucleotide polymorphisms (SNPs), allele-specific expression, and gene fusions, which contributes to the understanding of disease causal variants.

Retrotransposons are transposable elements which proliferate within eukaryotic genomes through a process involving reverse transcription. RNA-Seq can provide information about the transcription of endogenous retrotransposons that may influence the transcription of neighboring genes by various epigenetic mechanisms that lead to disease. Similarly, the potential for using RNA-Seq to understand immune-related disease is expanding rapidly due to the ability to dissect immune cell populations and to sequence T cell and B cell receptor repertoires from patients.

### Human and pathogen transcriptomes

RNA-Seq of human pathogens has become an established method for quantifying gene expression changes, identifying novel virulence factors, predicting antibiotic resistance, and unveiling host-pathogen immune interactions. A primary aim of this technology is to develop optimised infection control measures and targeted individualised treatment.

Transcriptomic analysis has predominantly focused on either the host or the pathogen. Dual RNA-Seq has been applied to simultaneously profile RNA expression in both the pathogen and host throughout the infection process. This technique enables the study of the dynamic response and interspecies gene regulatory networks in both interaction partners from initial contact through to invasion and the final persistence of the pathogen or clearance by the host immune system.

### Responses to environment

Transcriptomics allows identification of genes and pathways that respond to and counteract biotic and abiotic environmental stresses. The non-targeted nature of transcriptomics allows the identification of novel transcriptional networks in complex systems. For example, comparative analysis of a range of chickpea lines at different developmental stages identified distinct transcriptional profiles associated with drought and salinity stresses, including identifying the role of transcript isoforms of AP2-EREBP. Investigation of gene expression during biofilm formation by the fungal pathogen Candida albicans revealed a co-regulated set of genes critical for biofilm establishment and maintenance.

Transcriptomic profiling also provides crucial information on mechanisms of drug resistance. Analysis of over 1000 isolates of Plasmodium falciparum, a virulent parasite responsible for malaria in humans, identified that upregulation of the unfolded protein response and slower progression through the early stages of the asexual intraerythrocytic developmental cycle were associated with artemisinin resistance in isolates from Southeast Asia.

The use of transcriptomics is also important to investigate responses in the marine environment. In marine ecology, "stress" and "adaptation" have been among the most common research topics, especially related to anthropogenic stress, such as global change and pollution. Most of the studies in this area have been done in animals, although invertebrates have been underrepresented. One issue still is a deficiency in functional genetic studies, which hamper gene annotations, especially for non-model species, and can lead to vague conclusions on the effects of responses studied.

### Gene function annotation

All transcriptomic techniques have been particularly useful in identifying the functions of genes and identifying those responsible for particular phenotypes. Transcriptomics of Arabidopsis ecotypes that hyperaccumulate metals correlated genes involved in metal uptake, tolerance, and homeostasis with the phenotype. Integration of RNA-Seq datasets across different tissues has been used to improve annotation of gene functions in commercially important organisms (e.g. cucumber) or threatened species (e.g. koala).

Assembly of RNA-Seq reads is not dependent on a reference genome and so is ideal for gene expression studies of non-model organisms with non-existing or poorly developed genomic resources. For example, a database of SNPs used in Douglas fir breeding programs was created by de novo transcriptome analysis in the absence of a sequenced genome. Similarly, genes that function in the development of cardiac, muscle, and nervous tissue in lobsters were identified by comparing the transcriptomes of the various tissue types without use of a genome sequence. RNA-Seq can also be used to identify previously unknown protein coding regions in existing sequenced genomes.

#### A transcriptome based aging clock

Aging-related preventive interventions are not possible without personal aging speed measurement. The most up to date and complex way to measure aging rate is by using varying biomarkers of human aging is based on the utilization of deep neural networks which may be trained on any type of omics biological data to predict the subject's age. Aging has been shown to be a strong driver of transcriptome changes. Aging clocks based on transcriptomes have suffered from considerable variation in the data and relatively low accuracy. However an approach that uses temporal scaling and binarization of transcriptomes to define a gene set that predicts biological age with an accuracy allowed to reach an assessment close to the theoretical limit.

### Non-coding RNA

Transcriptomics is most commonly applied to the mRNA content of the cell. However, the same techniques are equally applicable to non-coding RNAs (ncRNAs) that are not translated into a protein, but instead have direct functions (e.g. roles in protein translation, DNA replication, RNA splicing, and transcriptional regulation). Many of these ncRNAs affect disease states, including cancer, cardiovascular, and neurological diseases.

## Transcriptome databases

Transcriptomics studies generate large amounts of data that have potential applications far beyond the original aims of an experiment. As such, raw or processed data may be deposited in public databases to ensure their utility for the broader scientific community. For example, as of 2018, the Gene Expression Omnibus contained millions of experiments.

Legend: NCBI – National Center for Biotechnology Information; EBI – European Bioinformatics Institute; DDBJ – DNA Data Bank of Japan; ENA – European Nucleotide Archive; MIAME – Minimum Information About a Microarray Experiment; MINSEQE – Minimum Information about a high-throughput nucleotide SEQuencing Experiment.

## See also

- omics
  - Genomics
  - Proteomics
  - Metabolomics
  - Venomics

Study of RNA transcripts


Sirius

Sirius is the brightest star in the night sky. Its name is derived from the Greek word Σείριος, or Seirios, meaning lit. 'glowing' or 'scorching'. The star is designated α Canis Majoris, Latinized to Alpha Canis Majoris, and abbreviated α CMa or Alpha CMa. With a visual apparent magnitude of −1.46, Sirius is almost twice as bright as Canopus, the next brightest star. Sirius is a binary star consisting of a main-sequence star of spectral type A0 or A1, termed Sirius A, and a faint white dwarf companion of spectral type DA2, termed Sirius B. The distance between the two varies between 8.2 and 31.5 astronomical units as they orbit every 50 years.

Sirius appears bright because of its intrinsic luminosity and its proximity to the Solar System. At a distance of 2.64 parsecs (8.6 ly), the Sirius system is one of Earth's nearest neighbours. Sirius is gradually moving closer to the Solar System; it is expected to increase in brightness slightly over the next 60,000 years to reach a peak magnitude of −1.68. Coincidentally, at about the same time, Sirius will take its turn as the southern Pole Star, around the year 66270. In that year, Sirius will come to within 1.6 degrees of the south celestial pole. This is due to precession and proper motion of Sirius itself which moves slowly in the SSW direction. So it will be visible from the southern hemisphere only.

After that time, its distance will begin to increase, and it will become fainter, but it will continue to be the brightest star in the Earth's night sky for approximately the next 210,000 years, at which point Vega, another A-type star that is intrinsically more luminous than Sirius, becomes the brightest star.

Sirius A is about twice as massive as the Sun () and has an absolute visual magnitude of +1.43. It is 25 times as luminous as the Sun, but has a significantly lower luminosity than other bright stars such as Canopus, Betelgeuse, or Rigel. The system is between 200 and 300 million years old. It was originally composed of two bright bluish stars. The initially more massive of these, Sirius B, consumed its hydrogen fuel and became a red giant before shedding its outer layers and collapsing into its current state as a white dwarf around 120 million years ago.

Sirius is colloquially known as the "Dog Star", reflecting its prominence in its constellation, Canis Major (the Greater Dog). The heliacal rising of Sirius marked the flooding of the Nile in Ancient Egypt and the "dog days" of summer for the ancient Greeks, while to the Polynesians, mostly in the Southern Hemisphere, the star marked winter and was an important reference for their navigation around the Pacific Ocean.

## Observational history

The brightest star seen from Earth, Sirius is recorded in some of the earliest astronomical records. Its displacement from the ecliptic causes its heliacal rising to be remarkably regular compared to other stars, with a period of almost exactly 365.25 days holding it constant relative to the solar year. This rising occurs at Cairo on 19 July (Julian), placing it just before the onset of the annual flooding of the Nile during antiquity. Owing to the flood's own irregularity, the extreme precision of the star's return made it important to the ancient Egyptians, who worshipped it as the goddess Sopdet (Ancient Egyptian: Spdt, "Triangle"; Greek: }, Sō̂this), guarantor of the fertility of their land.

The ancient Greeks observed that the appearance of Sirius as the morning star heralded the hot and dry summer and feared that the star caused plants to wilt, men to weaken, and women to become aroused. Owing to its brightness, Sirius would have been seen to twinkle more in the unsettled weather conditions of early summer. To Greek observers, this signified emanations that caused its malignant influence. Anyone suffering its effects was said to be "star-struck" (ἀστροβόλητος, astrobólētos). It was described as "burning" or "flaming" in literature. The season following the star's reappearance came to be known as the "dog days". The inhabitants of the island of Ceos in the Aegean Sea would offer sacrifices to Sirius and Zeus to bring cooling breezes and would await the reappearance of the star in summer. If it rose clear, it would portend good fortune; if it was misty or faint then it foretold (or emanated) pestilence. Coins retrieved from the island from the 3rd century BC feature dogs or stars with emanating rays, highlighting Sirius's importance.

The Romans celebrated the heliacal setting of Sirius around 25 April, sacrificing a dog, along with incense, wine, and a sheep, to the goddess Robigo so that the star's emanations would not cause wheat rust on wheat crops that year.

Bright stars were important to the ancient Polynesians for navigation of the Pacific Ocean. They also served as latitude markers; the declination of Sirius matches the latitude of the archipelago of Fiji at 17°S and thus passes directly over the islands each sidereal day. Sirius served as the body of a "Great Bird" constellation called Manu, with Canopus as the southern wingtip and Procyon the northern wingtip, which divided the Polynesian night sky into two hemispheres. Just as the appearance of Sirius in the morning sky marked summer in Greece, it marked the onset of winter for the Māori, whose name Takurua described both the star and the season. Its culmination at the winter solstice was marked by celebration in Hawaii, where it was known as Ka'ulua, "Queen of Heaven". Many other Polynesian names have been recorded, including Tau-ua in the Marquesas Islands, Rehua in New Zealand, and Ta'urua-fau-papa "Festivity of original high chiefs" and Ta'urua-e-hiti-i-te-tara-te-feiai "Festivity who rises with prayers and religious ceremonies" in Tahiti.

### Kinematics

In 1717, Edmond Halley discovered the proper motion of the hitherto presumed fixed stars after comparing contemporary astrometric measurements with those from the second century AD given in Ptolemy's Almagest. The bright stars Aldebaran, Arcturus and Sirius were noted to have moved significantly; Sirius had progressed about 30 arcminutes (about the diameter of the Moon) to the southwest.

In 1868, Sirius became the first star to have its velocity measured, the beginning of the study of celestial radial velocities. Sir William Huggins examined the spectrum of the star and observed a red shift. He concluded that Sirius was receding from the Solar System at about 40 km/s. Compared to the modern value of −5.5 km/s, this was an overestimate and had the wrong sign; the minus sign (−) means that it is approaching the Sun.

### Distance

In his 1698 book, Cosmotheoros, Christiaan Huygens estimated the distance to Sirius at 27,664 times the distance from the Earth to the Sun (about 0.437 light-year, translating to a parallax of roughly 7.5 arcseconds). There were several unsuccessful attempts to measure the parallax of Sirius: by Jacques Cassini (6 seconds); by some astronomers (including Nevil Maskelyne) using Lacaille's observations made at the Cape of Good Hope (4 seconds); by Piazzi (the same amount); using Lacaille's observations made at Paris, more numerous and certain than those made at the Cape (no sensible parallax); by Bessel (no sensible parallax).

Scottish astronomer Thomas Henderson used his observations made in 1832–1833 and South African astronomer Thomas Maclear's observations made in 1836–1837, to determine that the value of the parallax was 0.23 arcsecond, and error of the parallax was estimated not to exceed a quarter of a second, or as Henderson wrote in 1839, "On the whole we may conclude that the parallax of Sirius is not greater than half a second in space; and that it is probably much less." Astronomers adopted a value of 0.25 arcsecond for much of the 19th century. It is now known to have a parallax of nearly 0.4 arcseconds.

The Hipparcos parallax for Sirius is only accurate to about  light years, giving a distance of 8.6 light years. Sirius B is generally assumed to be at the same distance. Sirius B has a Gaia Data Release 3 parallax with a much smaller statistical margin of error, giving a distance of 8.709±0.005 light years, but it is flagged as having a very large value for astrometric excess noise, which indicates that the parallax value may be unreliable.

### Discovery of Sirius B

In a letter dated 10 August 1844, the German astronomer Friedrich Wilhelm Bessel deduced from changes in the proper motion of Sirius that it had an unseen companion. On 31 January 1862, American telescope-maker and astronomer Alvan Graham Clark first observed the faint companion, which is now called Sirius B, or affectionately "the Pup". This happened during testing of an 18.5-inch (470 mm) aperture great refractor telescope for Dearborn Observatory, which was one of the largest refracting telescope lenses in existence at the time, and the largest telescope in the United States. Sirius B's sighting was confirmed on 8 March with smaller telescopes.

The visible star is now sometimes known as Sirius A. Since 1894, some apparent orbital irregularities in the Sirius system have been observed, suggesting a third very small companion star, but this has never been confirmed. The best fit to the data indicates a six-year orbit around Sirius A and a mass of . This star would be five to ten magnitudes fainter than the white dwarf Sirius B, which would make it difficult to observe. Observations published in 2008 were unable to detect either a third star or a planet. An apparent "third star" observed in the 1920s is now believed to be a background object.

In 1915, Walter Sydney Adams, using a 60-inch (1.5 m) reflector at Mount Wilson Observatory, observed the spectrum of Sirius B and determined that it was a faint whitish star. This led astronomers to conclude that it was a white dwarf—the second to be discovered. The diameter of Sirius A was first measured by Robert Hanbury Brown and Richard Q. Twiss in 1959 at Jodrell Bank using their stellar intensity interferometer. In 2005, using the Hubble Space Telescope, astronomers determined that Sirius B has nearly the diameter of the Earth, 12,000 kilometres (7,500 mi), with a mass 102% of the Sun's.

### Colour controversy

Around the year 150 AD, Claudius Ptolemy of Alexandria, an ethnic Greek Egyptian astronomer of the Roman period, mapped the stars in Books VII and VIII of his Almagest, in which he used Sirius as the location for the globe's central meridian. He described Sirius as reddish, along with five other stars, Betelgeuse, Antares, Aldebaran, Arcturus, and Pollux, all of which are at present observed to be of orange or red hue. The discrepancy was first noted by amateur astronomer Thomas Barker, squire of Lyndon Hall in Rutland, who prepared a paper and spoke at a meeting of the Royal Society in London in 1760. The existence of other stars changing in brightness gave credibility to the idea that some may change in colour too; Sir John Herschel noted this in 1839, possibly influenced by witnessing Eta Carinae two years earlier. Thomas J.J. See resurrected discussion on red Sirius with the publication of several papers in 1892, and a final summary in 1926. He cited not only Ptolemy but also the poet Aratus, the orator Cicero, and general Germanicus all calling the star red, though acknowledging that none of the latter three authors were astronomers, the last two merely translating Aratus's poem Phaenomena. Seneca had described Sirius as being of a deeper red than Mars. Not all ancient observers saw Sirius as red. The 1st-century poet Marcus Manilius described it as "sea-blue", as did the 4th-century Avienius. It was the standard white star in ancient China, and multiple records from the 2nd century BC up to the 7th century AD all describe Sirius as white.

In 1985, German astronomers Wolfhard Schlosser and Werner Bergmann published an account of an 8th-century Lombardic manuscript, which contains De cursu stellarum ratio by St. Gregory of Tours. The Latin text taught readers how to determine the times of nighttime prayers from positions of the stars, and a bright star described as rubeola ("reddish") was claimed to be Sirius. The authors proposed this was further evidence Sirius B had been a red giant at the time. Other scholars replied that it was likely St. Gregory had been referring to Arcturus.

The possibility that stellar evolution of either Sirius A or Sirius B could be responsible for this discrepancy has been rejected by astronomers on the grounds that the timescale of thousands of years is much too short and that there is no sign of the nebulosity in the system that would be expected had such a change taken place. An interaction with a third star, to date undiscovered, has also been proposed as a possibility for a red appearance. Alternative explanations are either that the description as red is a poetic metaphor for ill fortune, or that the dramatic scintillations of the star when rising left the viewer with the impression that it was red. To the naked eye, it often appears to be flashing with red, white, and blue hues when near the horizon.

## Observation

With an apparent magnitude of −1.46, Sirius is the brightest star in the night sky, almost twice as bright as the second-brightest star, Canopus. From Earth, Sirius always appears dimmer than Jupiter and Venus, and at certain times also dimmer than Mercury and Mars. Sirius is visible from almost everywhere on Earth, except latitudes north of 73° N, and it does not rise very high when viewed from some northern cities (reaching only 13° above the horizon from Saint Petersburg). Because of its declination of roughly −17°, Sirius is a circumpolar star from latitudes south of 73° S. From the Southern Hemisphere in early July, Sirius can be seen in both the evening where it sets after the Sun and in the morning where it rises before the Sun. Along with Procyon and Betelgeuse, Sirius forms one of the three vertices of the Winter Triangle to observers in the Northern Hemisphere.

Sirius can be observed in daylight with the naked eye under the right conditions. Ideally, the sky should be very clear, with the observer at a high altitude, the star passing overhead, and the Sun low on the horizon. These observing conditions are more easily met in the Southern Hemisphere, owing to the southerly declination of Sirius.

The orbital motion of the Sirius binary system brings the two stars to a minimum angular separation of 3 arcseconds and a maximum of 11 arcseconds. At the closest approach, it is an observational challenge to distinguish the white dwarf from its more luminous companion, requiring a telescope with at least 300 mm (12 in) aperture and excellent seeing conditions. After a periastron occurred in 1994, the pair moved apart, making them easier to separate with a telescope. Apoastron occurred in 2019, but from the Earth's vantage point, the greatest observational separation will occur in 2023, with an angular separation of 11.333′′.

At a distance of 2.6 parsecs (8.6 ly), the Sirius system contains two of the eight nearest stars to the Sun, and it is the fifth closest stellar system to the Sun. This proximity is the main reason for its brightness, as with other near stars such as Alpha Centauri, Procyon and Vega and in contrast to distant, highly luminous supergiants such as Canopus, Rigel or Betelgeuse.(Note that Canopus may be a bright giant) It is still around 25 times more luminous than the Sun. The closest large neighbouring star to Sirius is Procyon, 1.61 parsecs (5.24 ly) away. The Voyager 2 spacecraft, launched in 1977 to study the four giant planets in the Solar System, is expected to pass within 4.3 light-years (1.3 pc) of Sirius in approximately 296,000 years.

## Stellar system

Sirius is a binary star system consisting of two white stars orbiting each other with a separation of about 20 AU (roughly the distance between the Sun and Uranus) and a period of 50.1 years. The brighter component, termed Sirius A, is a main-sequence star of spectral type early A, with an estimated surface temperature of 9,940 K. Its companion, Sirius B, is a star that has already evolved off the main sequence and become a white dwarf. Currently 10,000 times less luminous in the visual spectrum, Sirius B was once the more massive of the two. The age of the system has been estimated at around 230 million years. Early in its life, it is thought to have been two bluish-white stars orbiting each other in an elliptical orbit every 9.1 years. The system emits a higher than expected level of infrared radiation, as measured by IRAS space-based observatory. This might be an indication of dust in the system, which is considered somewhat unusual for a binary star. The Chandra X-ray Observatory image shows Sirius B outshining its partner as an X-ray source.

In 2015, Vigan and colleagues used the VLT Survey Telescope to search for evidence of substellar companions, and were able to rule out the presence of giant planets 11 times more massive than Jupiter at 0.5 AU distance from Sirius A, 6–7 times the mass of Jupiter at 1–2 AU distance, and down to around 4 times the mass of Jupiter at 10 AU distance. Similarly, Lucas and colleagues did not detect any companions around Sirius B.

### Sirius A

Sirius A, also known as the Dog Star, has a mass of . The radius of this star has been measured by an astronomical interferometer, giving an estimated angular diameter of 5.936±0.016 mas. The projected rotational velocity is a relatively low 16 km/s, which does not produce any significant flattening of its disk. This is at marked variance with the similar-sized Vega, which rotates at a much faster 274 km/s and bulges prominently around its equator. A weak magnetic field has been detected on the surface of Sirius A.

Stellar models suggest that the star formed during the collapsing of a molecular cloud and that, after 10 million years, its internal energy generation was derived entirely from nuclear reactions. The core became convective and used the CNO cycle for energy generation. It is calculated that Sirius A will have completely exhausted the store of hydrogen at its core within a billion (109) years of its formation, and will then evolve away from the main sequence. It will pass through a red giant stage and eventually become a white dwarf.

Sirius A is classed as a type Am star, because the spectrum shows deep metallic absorption lines, indicating an enhancement of its surface layers in elements heavier than helium, such as iron. The spectral type has been reported as A0mA1 Va, which indicates that it would be classified as A1 from hydrogen and helium lines, but A0 from the metallic lines that cause it to be grouped with the Am stars. When compared to the Sun, the proportion of iron in the atmosphere of Sirius A relative to hydrogen is given by $\textstyle\ \left[\frac{\ce{Fe}}{\ce{H}}\right] = 0.5\ ,$ meaning iron is 316% as abundant as in the Sun's atmosphere. The high surface content of metallic elements is unlikely to be true of the entire star; rather the iron-peak and heavy metals are radiatively levitated towards the surface.

### Sirius B

Sirius B, also known as the Pup Star, is one of the most massive white dwarfs known. With a mass of , it is almost double the average. This mass is packed into a volume roughly equal to the Earth's. The current surface temperature is 25,200 K. Because there is no internal heat source, Sirius B will steadily cool as the remaining heat is radiated into space over the next two billion years or so.

A white dwarf forms after a star has evolved from the main sequence and then passed through a red giant stage. This occurred when Sirius B was less than half its current age, around 120 million years ago. The original star had an estimated and was a B-type star (most likely B5V for ) when it was still on the main sequence, potentially burning around 600-1200 times more luminous than the sun. While it passed through the red giant stage, Sirius B may have enriched the metallicity of its companion, explaining the very high metallicity of Sirius A.

This star is primarily composed of a carbon–oxygen mixture that was generated by helium fusion in the progenitor star. This is overlaid by an envelope of lighter elements, with the materials segregated by mass because of the high surface gravity. The outer atmosphere of Sirius B is now almost pure hydrogen—the element with the lowest mass—and no other elements are seen in its spectrum.

### Apparent third star

Since 1894, irregularities have been tentatively observed in the orbits of Sirius A and B with an apparent periodicity of 6–6.4 years. A 1995 study concluded that such a companion likely exists, with a mass of roughly 0.05 solar mass—a small red dwarf or large brown dwarf, with an apparent magnitude of more than 15, and less than 3 arcseconds from Sirius A.

More recent (and accurate) astrometric observations by the Hubble Space Telescope ruled out the existence of such a Sirius C entirely. The 1995 study predicted an astrometric movement of roughly 90 mas (0.09 arcsecond), but Hubble was unable to detect any location anomaly to an accuracy of 5 mas (0.005 arcsec). This ruled out any objects orbiting Sirius A with more than 0.033 solar mass (35 Jupiter masses) orbiting in 0.5 years, and 0.014 (15 Jupiter masses) in 2 years. The study was also able to rule out any companions to Sirius B with more than 0.024 solar mass (25 Jupiter masses) orbiting in 0.5 year, and 0.0095 (10 Jupiter masses) orbiting in 1.8 years. Effectively, there are almost certainly no additional bodies in the Sirius system larger than a small brown dwarf or large exoplanet.

### Star cluster membership

In 1909, Ejnar Hertzsprung was the first to suggest that Sirius was a member of the Ursa Major Moving Group, based on his observations of the system's movements across the sky. The Ursa Major Group is a set of 220 stars that share a common motion through space. It was once a member of an open cluster, but has since become gravitationally unbound from the cluster. Analyses in 2003 and 2005 found Sirius's membership in the group to be questionable: the Ursa Major Group has an estimated age of 500 ± 100 million years, whereas Sirius, with metallicity similar to the Sun's, has an age that is only half this, making it too young to belong to the group. Sirius may instead be a member of the proposed Sirius Supercluster, along with other scattered stars such as Beta Aurigae, Alpha Coronae Borealis, Beta Crateris, Beta Eridani and Beta Serpentis. This would be one of three large clusters located within 500 light-years (150 pc) of the Sun. The other two are the Hyades and the Pleiades, and each of these clusters consists of hundreds of stars.

### Distant star cluster

In 2017, a massive star cluster was discovered only 10 arcminutes from Sirius, making the two appear to be visually close to one other when viewed from the point of view of the Earth. It was discovered during a statistical analysis of Gaia data. The cluster is over a thousand times further away from us than the star system, but given its size it still appears at magnitude 8.3 .

## Etymology

The proper name "Sirius" comes from the Latin Sīrius, from the Ancient Greek Σείριος (Seirios, "glowing" or "scorcher"). The Greek word itself may have been imported from elsewhere before the Archaic period, one authority suggesting a link with the Egyptian god Osiris. The name's earliest recorded use dates from the 7th century BC in Hesiod's poetic work Works and Days. In 2016, the International Astronomical Union organized a Working Group on Star Names (WGSN) to catalog and standardize proper names for stars. The WGSN's first bulletin of July 2016 included a table of the first two batches of names approved by the WGSN, which included Sirius for the star α Canis Majoris A. It is now so entered in the IAU Catalog of Star Names.

Sirius has over 50 other designations and names attached to it. In Geoffrey Chaucer's essay Treatise on the Astrolabe, it bears the name Alhabor and is depicted by a hound's head. This name is widely used on medieval astrolabes from Western Europe. In Sanskrit it is known as Mrgavyadha "deer hunter", or Lubdhaka "hunter". As Mrgavyadha, the star represents Rudra (Shiva). The star is referred to as Makarajyoti in Malayalam and has religious significance to the pilgrim center Sabarimala. In Scandinavia, the star has been known as Lokabrenna ("burning done by Loki", or "Loki's torch"). In the astrology of the Middle Ages, Sirius was a Behenian fixed star, associated with beryl and juniper. Its astrological symbol was listed by Heinrich Cornelius Agrippa.

## Cultural significance

Many cultures have historically attached special significance to Sirius, particularly in relation to dogs. It is often colloquially called the "Dog Star" as the brightest star of Canis Major, the "Great Dog" constellation. Canis Major was classically depicted as Orion's dog. The Ancient Greeks thought that Sirius's emanations could affect dogs adversely, making them behave abnormally during the "dog days", the hottest days of the summer. The Romans knew these days as dies caniculares, and the star Sirius was called Canicula, "little dog". The excessive panting of dogs in hot weather was thought to place them at risk of desiccation and disease. In extreme cases, a foaming dog might have rabies, which could infect and kill humans they had bitten. Homer, in the Iliad, describes the approach of Achilles toward Troy in these words:

> > Sirius rises late in the dark, liquid sky On summer nights, star of stars, Orion's Dog they call it, brightest Of all, but an evil portent, bringing heat And fevers to suffering humanity.

In a little-attested Greek myth, the star-god that personified Sirius fell in love with a fertility goddess named Opora, but he was unable to have her. Thus he began to burn hot, making humans suffer, who prayed to the gods. The god of the north wind, Boreas, solved the problem by ordering his sons to deliver Opora to Sirius, while he cooled down the earth with blasts of his own cold wind.

In Iranian mythology, especially in Persian mythology and in Zoroastrianism, the ancient religion of Persia, Sirius appears as Tishtrya and is revered as the rain-maker divinity (Tishtar of New Persian poetry). Beside passages in the sacred texts of the Avesta, the Avestan language Tishtrya followed by the version Tir in Middle and New Persian is also depicted in the Persian epic Shahnameh of Ferdowsi. Because of the concept of the yazatas, powers which are "worthy of worship", Tishtrya is a divinity of rain and fertility and an antagonist of apaosha, the demon of drought. In this struggle, Tishtrya is depicted as a white horse.

In Chinese astronomy Sirius is known as the star of the "celestial wolf" ( Chinese romanization: Tiānláng; Japanese romanization: Tenrō; Korean and romanization: 천랑 /Cheonrang) in the Mansion of Jǐng (井宿). Many nations among the indigenous peoples of North America also associated Sirius with canines; the Seri and Tohono Oʼodham of the southwest note the star as a dog that follows mountain sheep, while the Blackfoot called it "Dog-face". The Cherokee paired Sirius with Antares as a dog-star guardian of either end of the "Path of Souls". The Pawnee of Nebraska had several associations; the Wolf (Skidi) tribe knew it as the "Wolf Star", while other branches knew it as the "Coyote Star". Further north, the Alaskan Inuit of the Bering Strait called it "Moon Dog".

Several cultures also associated the star with a bow and arrows. The ancient Chinese visualized a large bow and arrow across the southern sky, formed by the constellations of Puppis and Canis Major. In this, the arrow tip is pointed at the wolf Sirius. A similar association is depicted at the Temple of Hathor in Dendera, where the goddess Satet has drawn her arrow at Hathor (Sirius). Known as "Tir", the star was portrayed as the arrow itself in later Persian culture.

Sirius is mentioned in Surah, An-Najm ("The Star"), of the Qur'an, where it is given the name الشِّعْرَى (transliteration: aš-ši'rā or ash-shira; the leader). The verse is: "وأنَّهُ هُوَ رَبُّ الشِّعْرَى", "That He is the Lord of Sirius (the Mighty Star)." (An-Najm:49) Ibn Kathir said in his commentary "that it is the bright star, named Mirzam Al-Jawza' (Sirius), which a group of Arabs used to worship". The alternate name Aschere, used by Johann Bayer, is derived from this.

In theosophy, it is believed the Seven Stars of the Pleiades transmit the spiritual energy of the Seven Rays from the Galactic Logos to the Seven Stars of the Great Bear, then to Sirius. From there is it sent via the Sun to the god of Earth (Sanat Kumara), and finally through the seven Masters of the Seven Rays to the human race.

The midnight culmination of Sirius in the northern hemisphere coincides with the beginning of the New Year of the Gregorian Calendar during the decades around the year 2000. Over the years, its midnight culmination moves slowly, owing to the combination of the star's proper motion and the precession of the equinoxes. At the time of the introduction of the Gregorian calendar in the year 1582, its culmination occurred 17 minutes before midnight into the new year under the assumption of a constant motion. According to Richard Hinckley Allen its mightnight culmination was celebrated at the Temple of Demeter at Eleusis.

### Dogon

The Dogon people are an ethnic group in Mali, West Africa, reported by some researchers to have traditional astronomical knowledge about Sirius that would normally be considered impossible without the use of telescopes. According to Marcel Griaule, they knew about the fifty-year orbital period of Sirius and its companion prior to western astronomers.

Doubts have been raised about the validity of Griaule and Dieterlein's work. In 1991, anthropologist Walter van Beek concluded about the Dogon, "Though they do speak about sigu tolo [which is what Griaule claimed the Dogon called Sirius] they disagree completely with each other as to which star is meant; for some it is an invisible star that should rise to announce the sigu [festival], for another it is Venus that, through a different position, appears as sigu tolo. All agree, however, that they learned about the star from Griaule." According to Noah Brosch cultural transfer of relatively modern astronomical information could have taken place in 1893, when a French expedition arrived in Central West Africa to observe the total eclipse on 16 April.

### Serer religion

In the religion of the Serer people of Senegal, the Gambia and Mauritania, Sirius is called Yoonir from the Serer language (and some of the Cangin language speakers, who are all ethnically Serers). The star Sirius is one of the most important and sacred stars in Serer religious cosmology and symbolism. The Serer high priests and priestesses (Saltigues, the hereditary "rain priests") chart Yoonir in order to forecast rainfall and enable Serer farmers to start planting seeds. In Serer religious cosmology, it is the symbol of the universe.

### Modern significance

Sirius features on the coat of arms of Macquarie University, and is the name of its alumnae journal. Seven ships of the Royal Navy have been called HMS Sirius since the 18th century, with the first being the flagship of the First Fleet to Australia in 1788. The Royal Australian Navy subsequently named a vessel in honor of the flagship. American vessels include the USNS Sirius (T-AFS-8) as well as a monoplane model—the Lockheed Sirius, the first of which was flown by Charles Lindbergh. The name was also adopted by Mitsubishi Motors as the Mitsubishi Sirius engine in 1980. The name of the North American satellite radio company CD Radio was changed to Sirius Satellite Radio in November 1999, being named after "the brightest star in the night sky". Sirius is one of the 27 stars on the flag of Brazil, where it represents the state of Mato Grosso.

Composer Karlheinz Stockhausen, who wrote a piece called Sirius, is claimed to have said on several occasions that he came from a planet in the Sirius system. To Stockhausen, Sirius stood for "the place where music is the highest of vibrations" and where music had been developed in the most perfect way.

Sirius has been the subject of poetry. Dante and John Milton reference the star, and it is the "powerful western fallen star" of Walt Whitman's "When Lilacs Last in the Dooryard Bloom'd", while Tennyson's poem The Princess describes the star's scintillation:

> > ...the fiery Sirius alters hue And bickers into red and emerald.

## See also

- List of stars in Canis Major
- List of nearest stars and brown dwarfs
  - Historical brightest stars
- Sirius in fiction

Brightest star in the night sky, in the constellation Canis Major

Astronomical objects known since antiquity