1. Introduction {#s0005}
===============

The brewing process is probably the most ancient type of biotechnology. There is evidence that the production and consumption of beer began in Egypt in the Early Dynastic Period (5500--3100 BCE) [@bb0005]. The first fermentation process was termed "high" because yeast floats to the top of the tank at a temperature between 15 °C and 25 °C during production of ale beer with the yeast *Saccharomyces cerevisiae*. Lager yeast did not emerge until the 15th century. This yeast is capable of fermenting at a temperature lower than 10 °C and flocculates at the bottom of the tank. The fermentation is followed by a maturation process called "lagering" (Masschelein, 1986).

The *Saccharomyces sensu stricto* complex includes six parented species: *S. cerevisiae*, *Saccharomyces bayanus*, *Saccharomyces cariocanus*, *Saccharomyces kudriavzevii*, *S. mikatae* and *S. paradoxus* [@bb0010]; however, it has been observed that lager-brewing yeast is a hybrid species of two combined genomes of *S. eubayanus* and *S. cerevisiae* [@bb0015], [@bb0020], [@bb0025], [@bb0030], [@bb0035], [@bb0040], [@bb0045], [@bb0050]. This provides an important source of chromosomal rearrangements, leading to the gene number and the size of the complete genome [@bb0055], [@bb0060], [@bb0065], [@bb0070]. It has been proposed and recently demonstrated that lager yeast is the product of two independent hybridization events that can be divided into two groups: Saaz and Frohberg, or group I and group II, respectively [@bb0075], [@bb0080], [@bb0085], [@bb0090], [@bb0095].

With the use of next generation sequencing (NGS) technologies, such as the Illumina Platform, 40,175 prokaryote and eukaryotes genomes have been reported, including 210 different strains of the *Saccharomyces* complex (<http://www.ncbi.nlm.nih.gov/genome/browse/> - revised July 22, 2015).

To obtain a higher level of understanding of the sequenced organism, the data obtained from NGS has been assembled and annotated.

The annotation process consists of identifying the biological characteristics from sequences of the assembly. This can be performed through gene prediction and homologous sequence alignment [@bb0100], [@bb0105], [@bb0110].

Here, we present the analysis of the lager yeast genome *Saccharomyces* sp. strain 790 and its comparison with *S. eubayanus* and *S. cerevisiae* S288c. This study provides information about the genome structure of *Saccharomyces* sp. strain 790.

2. Materials and methods {#s0010}
========================

2.1. Strains and sequences {#s0015}
--------------------------

The brewing yeast *Saccharomyces* sp. strain 790 and a reference sequence of 76 scaffolds from *S. eubayanus* were obtained from the yeast collection of Cervecería Cuauhtémoc Moctezuma S.A. de C.V. The *S. cerevisiae* S288c reference genome sequence was retrieved from the yeast genome database ([www.yeastgenome.org](http://www.yeastgenome.org){#ir0015}).

2.2. Sequencing and genome assembly {#s0020}
-----------------------------------

The brewing yeast genome was sequenced using the FLX 454 Titanium (Roche) and MiSeq (Illumina) massive sequencing platforms according to the manufacturer\'s protocols. We obtained 0.8 million reads from FLX 454 Titanium (454 Life Sciences, Branfort, CT) with an average size of 400 bp; 6 million pair-end reads from Illumina (Illumina, San Diego, CA) with an average size of 150 bp; 5 million mate-pair reads from Illumina with an insert size of 350 bp and a size of 101 × 2 bp; and 11.7 million mate-pair reads from Illumina with an insert of 8 kb and a size of 51 × 2 bp. Approximately 454 Illumina pair-end reads were assembled with a Newbler DeNovo Assembler (Roche). Contigs were then processed using SSPACE 1.0 software (Boetzer et al. 2011). This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession LSMH00000000. The version described in this paper is version LSMH01000000.

The sequencing quality data were analyzed with FastQC 0.10.1 software with a value ≥ Q30 [@bb0115].

Likewise, alignments were made against reference sequences (*S. cerevisiae* S288c and *S. eubayanus*) with the MUMmer 3.23 software package [@bb0120].

2.3. Genome annotation {#s0025}
----------------------

The bioinformatics analysis for the annotation was performed with MAKER v2.31.8 software (University of Utah). MAKER is an integrative tool that yields a putative position of the genes. [Fig. 1](#f0005){ref-type="fig"} depicts the annotation steps [@bb0110].

3. Results and discussion {#s0030}
=========================

The read assembly yielded 133 scaffolds with a \~ 70 × depth and a N50 of 568,800 bp, suggesting a complete genome size of \~ 22.7 Mbp ([Table 1](#t0005){ref-type="table"}), similar to previous reports of other lager beer yeasts [@bb0055], [@bb0075], [@bb0125], [@bb0130]. Approximately 65/133 scaffolds had a size \> 10 kbp, which represents 99.667% of the assembled genome ([Table 2](#t0010){ref-type="table"}). [Table 3](#t0015){ref-type="table"} shows a comparison of the assembly level of the sequenced genomes of the *Saccharomyces* species (as of August 2015). It also shows a similar genome size compared to other brewing yeasts [@bb0055], [@bb0130]. The alignments against the reference genome, *S. cerevisiae* S288C, assigned scaffolds to each of its 16 chromosomes, and some scaffolds covered different portions of more than one chromosome; for example, scaffold01 (SF01) aligns with two chromosomes: a small portion with chromosome 1 and with chromosome 12. Scaffold17 (SF17) aligns with chromosomes 4 and 5 ([Fig. 2](#f0010){ref-type="fig"}).

The estimated size matches the previous and known information; this is due to the presence of 16 chromosomes of the *S. cerevisiae* sub-genome and 16 of *S. eubayanus*. This suggests an overall estimation of 32 chromosomes without considering ploidy. Likewise, its size is close to the sum of the aforementioned genomes (\~ 12 Mbp each). This observation is consistent with the previously reported data by Nakao et al. (2009), Borneman et al. (2011) and Walther et al. (2014), who reported the sequence and assembly of the lager brewing yeast genomes *Saccharomyces carlsbergensis* (78 scaffolds, 29 chromosomes with a 19.5 Mbp length), and *Saccharomyces pastorianus* Weihenstephan 34/70 (985 scaffolds, \~ 29 chromosomes, and 22.9 Mbp).

The annotation yielded 9939 CDS and a gff file with their locations in the scaffolds of the assembly ([Fig. 3](#f0015){ref-type="fig"}). The protein and transcript sequences, were subjected to a local alignment with the Blast tool [@bb0135] against a local database using the sequence of *S. cerevisiae* S288C as a reference. The transcripts were considered to be genes because previous reports showed that only approximately 5% of the yeast genome contains introns [@bb0140], [@bb0145].

The scaffolds were classified using the results obtained from Blastn according to the mean identity percentage in all of the genes contained in the same scaffold, as follows (Table 4):

%Id \> 99.0% and E value \< 10^− 6^ = scaffold belongs to *S. cerevisiae.*

%Id \< 90.0% and E value \< 10^− 6^ = scaffold does not belong to *S. cerevisiae.*

99.0% \> %Id \> 90.0% and E value \< 10^− 6^ = hybrid scaffold.

Our identity criterion was validated by subjecting the gene sequences from *S. cerevisiae* S288C to a local alignment against *S. eubayanus*, and we found that the %Id between these strains was \< 90% ([Supplementary Table S1](#ec0005){ref-type="supplementary-material"}) and the average size of the CDS was 1550 bp. Approximately 96.8% of the genome was annotated; 53.93% corresponded to *S. cerevisiae*, 42.86% were non-*cerevisiae* and 3.20% remained un-annotated [Fig. 4](#f0020){ref-type="fig"} ([Supplementary Table S2](#ec0010){ref-type="supplementary-material"}).

4. Conclusions {#s0035}
==============

From the findings in this work, it can be concluded that *Saccharomyces* sp. 790 is a hybrid between *S. cerevisiae* and *S. eubayanus.* Its nuclear genome consists of approximately 32 chromosomes, 16 of which correspond to the *S. cerevisiae* genome and 16 to the *S. eubayanus* genome, without considering ploidy. A total of 133 scaffolds were obtained in the last version of the assembly. Nine scaffolds presented continuous translocations (scaffolds 1, 4, 6, 23 for the *S. cerevisiae* sub-genome and 26, 11, 17, 22 and 32 for the *S. eubayanus* sub-genome), which indicate homologous recombination events. One scaffold presented a possible recombination event (scaffold 3). Data on the chromosome number and size, as well as the number of scaffolds obtained, are consistent with previous reports on lager yeast [@bb0030], [@bb0075], [@bb0080]. The next step is to improve the assembly with physical mapping techniques.

The following are the supplementary data related to this article.Supplementary Table S1Comparative analysis of *Saccharomyces cerevisiae* S288C and *Saccharomyces eubayanus*.Supplementary Table S1Supplementary Table S2*Saccharomyces* sp. 790 and *Saccharomyces cerevisiae* comparative results.Supplementary Table S2

This work was supported, in part, by the Consejo Nacional de Ciencia y Tecnología (CONACYT) of México Grant number: 212773. P. M. De León-Medina and R. Elizondo-González were recipients of a fellowship from CONACYT.

![Maker\'s annotation pipeline. In step 1, the repetitive sequences are identified and masked; proteins and ESTs are aligned; and using this information, a gene prediction model is made and the structural annotation is completed. Those models are then submitted to a local alignment in Blast for functional annotation.](gr1){#f0005}

![Alignment dot plot of *Saccharomyces* sp. 790 versus *S. cerevisiae* S288C.](gr2){#f0010}

![Visualization of annotations with IGV Tools v2.3 (<http://www.broadinstitute.org/software/igv/home>). Scaffold 01 alignments and predictions from the Maker\'s annotation.](gr3){#f0015}

![Schematic representation of *Saccharomyces sp.* 790 annotations. *S. cerevisiae*, *S. eubayanus* and non-annotated proportions are represented in blue, orange and gray, respectively.](gr4){#f0020}

###### 

Assembly of *Saccharomyces* sp. 790.

Table 1

  Assembled reads              17,034,361
  ---------------------------- ------------
  Depth                        \~ 70 ×
  Estimated genome size (bp)   22,741,276
  Number of scaffolds          133
  Average scaffold size (bp)   170,987
  Largest scaffold (bp)        1,404,408
  N50                          568,800
  'Ns'                         399,699

###### 

Size range among scaffolds.

Table 2

  Size level        Number of scaffolds   Bp           Whole genome proportion (%)   Cumulative % of the whole genome
  ----------------- --------------------- ------------ ----------------------------- ----------------------------------
  0:999             47                    31,168       0.137                         0.137
  1 k:9999          21                    44,610       0.196                         0.333
  10 k: 99,999      18                    1,063,816    4.678                         5.011
  100 k 199, 999    11                    1,608,982    7.075                         12.086
  200 k: 299,999    6                     1,528,906    6.723                         18.809
  300 k: 399,999    6                     2,092,805    9.203                         28.01
  400 k: 499, 999   4                     1,700,637    7.478                         35.490
  500 k: 599, 999   7                     3,759,004    16.529                        52.020
  600 k: 699,999    5                     3,213,208    14.129                        66.149
  700 k: 799,999    1                     712, 480     3.133                         69.282
  800 k: 899,999    1                     806,158      3.545                         72.827
  900 k: 999,999    5                     4,775,094    20.997                        93.824
  1;404,408         1                     1,404,408    6.176                         100
  Total             133                   22,741,276                                 

###### 

Sequenced genome assembly level of *Saccharomyces* species.

Table 3

  Organism                                        Finished genome   Number of chromosomes   Number of scaffolds   Number of contigs   Size (Mpb)
  ----------------------------------------------- ----------------- ----------------------- --------------------- ------------------- ------------
  *S. cerevisiae*                                 ✓                 16                      17                    --                  12.16
  *S. kudriavzevii*                               ✗                 16                      2054                  --                  11.19
  *S. pastorianus*                                ✗                 --                      --                    2425                24.21
  *S. paradoxus*                                  ✗                 16                      --                    832                 11.87
  *S. mikatae*                                    ✗                 16                      --                    1648                11.47
  *S. bayanus*                                    ✗                 16                      --                    586                 11.87
  *S. boulardii*                                  ✗                 16                      48                    --                  11.64
  *S. arboricola*                                 ✓                 16                      35                    --                  11.62
  *S. uvarum*                                     ✗                 --                      --                    3985                11.60
  *S. carlsbergensis*                             ✗                 29                      77                    --                  19.37
  *S. cerevisiae* - *S. kudriavzevii*             ✗                 --                      60                    419                 23.37
  *S. pastorianus*---*S*. *weihenstephan* 34/70   ✗                 --                      --                    1358                22.96
  **790**                                         ✗                 **\~ 32**               **133**               --                  **22.74**
