End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries
© Suetsugu et al; licensee BioMed Central Ltd. 2007
Received: 14 February 2007
Accepted: 07 September 2007
Published: 07 September 2007
We performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an Eco RI- and a Bam HI-digested library) and conducted an in silico analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (Bombyx mori).
More than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in Bam HI BESs, whereas DNA-type elements were abundant in Eco RI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of ≥ 99%. About 14% of Eco RI BESs and about 8% of Bam HI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions.
As a result of this characterization, the identified BESs will be a valuable resource for genomic research on Bombyx mori, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are DE283657–DE378560.
The silkworm (Bombyx mori) has been domesticated for more than 5000 years because of the industrial importance of sericulture. Besides being used for silk production, the silkworm is also an effective host for the production of recombinant proteins and biomaterials [1–3]. It is also an important model organism of the Lepidoptera, the insect order that includes the majority of serious agricultural pests. Therefore, the accumulation of silkworm genome resources will be helpful for both the control of agricultural pests and the development of the silkworm as an industrial-scale resource of biomaterials or bioreactors.
In silkworm, two individual whole-genome shotgun (WGS) projects have been carried out, and draft genomic sequences with 3× or 5.9× coverage have been generated [4, 5]. Databases of expressed sequence tags (ESTs) and a single nucleotide polymorphism linkage map have also been released [6, 7]. Bacterial artificial chromosomes (BACs) , as well as fosmids , also constitute important genomic resources. The main advantage of BACs, compared with yeast artificial chromosomes  or cosmids  is their higher stability, simplicity of construction and screening, low frequency of chimeric clones, and ease of DNA isolation. Therefore, BACs are one of the main tools used for high-throughput genomic studies, including for sequence-tagged connector (STC) strategies, BAC-based physical maps, and DNA fingerprinting, in various species [12–26].
BAC end sequences (BESs), single-pass sequence reads from each end of a BAC clone, are a powerful tool that enhances the value of BACs as a genomic resource [27–31]. We conducted large-scale BAC end-sequencing of two silkworm BAC libraries, the RPCI-96 Bombyx mori Silkworm P50 BAC Library  and the Texas A&M BAC Library , and characterized 94904 BESs.
Summary of two bacterial artificial chromosome (BAC) libraries
Eco RI-digested library
Bam HI-digested library
Number of clones
36000 (96 × 384 wells)
21120 (55 × 384 wells)
Mean insert size (kbp)
p50T (mixed insects)
p50T (mixed insects)
Characteristics of the two groups of BAC end sequences (BESs)
Eco RI BESs
Bam HI BESs
Number of sequences
Average read length (bp)
Minimum read length (bp)
Maximum read length (bp)
Total bases (bp)
GC content (%)
Percentage of paired-end clones (%)
Repeat analysis of BESs
Distribution of interspersed repeat DNA sequences within both BAC end sequences (BESs) in different repeat classes
Eco RI BESs
Bam HI BESs
To find novel repeat sequences in the BESs, we analyzed the repeat-masked BESs with RECON (version 1.05) , which automatically identifies de novo repeats. Only detected repeat families with 50 or more members were retained for further analysis. As a result, 31 and 15 repeat families with 50 or more members were detected in the Eco RI and Bam HI BESs, respectively. We then used BLASTX  to compare each repeat sequence against the nr (non-redundant protein) database, and found that 34.0% of the sequences had similarity to TE-related proteins. We used representative sequences of the repeat families for a BLAST search of silkworm whole-genome shotgun (WGS) data  to confirm whether they were really dispersed throughout the genome. The estimated copy number ranged from 9 to 2431; therefore, a large proportion of the detected sequences could be regarded as repetitive. However, a few sequences showed a much lower copy number than that estimated by RECON. It was recently reported that the great majority of silkworm transposon insertions are 5' -truncated, so most of the detected repeat sequences may be ''transposon fossils'' with no activity . Further analysis of the detected sequences might reveal novel transposons in silkworm.
BLAST search against whole-genome shotgun data
All BESs were used as queries in a BLAST similarity search of the two available sets of WGS data: the WGS data set deposited by the Silkworm Genomic Research Program  (abbreviated as "SGP data" in this paper) and the data set deposited by the Beijing Genomics Institute  (abbreviated as "BGI data"). In this search, the expectation value (-e option, a probability cutoff value) was set to 1e-5 and the -b option (number of database sequences to show) was set to 1000.
The majority of BESs with a match were BES+, having only one match in each WGS data set. In addition, the percentage of ''multi-match'' Eco RI BESs (BES++ in Fig. 2) was lower than that of multi-match Bam HI BESs. We inferred each BES+ to be a unique region-derived sequence, and BES++ to be likely derived from repetitive sequences. We defined ''unique paired-end clones'' as paired-end clones showing a single match at each BES. A BLAST search of SGP data using the BESs as queries identified 8104 unique paired-end clones in the Eco RI library and 2778 among the Bam HI BESs. Similarly, a BLAST search of the BGI data yielded 8878 paired-end clones in the Eco RI BAC library and 3102 in the Bam HI BAC library. A total of 4757 unique paired-end clones among the Eco RI BESs, and 1482 among the Bam HI BESs, were common to both WGS data sets.
BES clustering and coding region composition
Summary of clustering results
Cluster size d
Eco RI BAC ends (%)
Bam HI BAC ends (%)
d = 1 (singleton)
4 > d ≥ 2
8 > d ≥ 4
16 > d ≥ 8
32 > d ≥ 16
64 > d ≥ 32
128 > d ≥ 64
d ≥ 128
Each representative sequence was then searched against the GenBank nr database (BLASTX, with the e-value set to 1e-05) to investigate the percentage of BESs containing protein-coding regions. As a result, 8068 clusters (20.2%) of Eco RI BESs had similarity to proteins in the database, compared with 6905 clusters (28.2%) of Bam HI BESs. For Eco RI BESs, most of the hit proteins were from Bombyx mori (53.8% of the clusters with similarity to proteins in the database), Anopheles gambiae (6.8%), Apis mellifera (4.0%), Drosophila melanogaster (3.2%), or Bos taurus (1.6%), whereas in the case of Bam HI BESs, most of the hit proteins were from Bombyx mori (68.4%), Anopheles gambiae (11.7%), Apis mellifera (8.5%), Drosophila melanogaster (2.4%), or Bos taurus (2.5%). The majority of large clusters showed similarity to TE-related proteins.
Bam HI BESs contained more repetitive sequences than Eco RI BESs. In particular, the two groups of BESs contrasted with regard to the abundance of LINEs. The GC bias of Bam HI may be main factor accounting for this difference because the GC% of Bam HI recognition sites was relatively close to the estimated GC% of protein coding DNA of the silkworm genome. This inference is further supported by the fact that the LINEs in the repeat sequences library had Bam HI recognition sites at average intervals of 2.0 kbp, whereas the average interval between Eco RI recognition sites was 3.0 kbp. These results indicate that the use of multiple BAC libraries constructed with different restriction enzymes can increase the genome representation .
The GC content of the masked region, especially the LINEs-derived region, was much higher than that of the unmasked region (Table 3). Conversely, the GC% of the DNA transposons-derived region was similar to that of the coding region. To confirm the GC-richness of silkworm LINEs, we calculated the GC content of each type of transposable element in the repeat sequences library and found that the median GC content of DNA-type elements (67 sequences), long terminal repeat (LTR) elements (30 sequences), LINEs (69 sequences), and short interspersed elements (SINEs) (26 sequences) was 39.1%, 43.7%, 51.9%, and 46.6%, respectively. Thus, the GC% of silkworm LINEs was rather higher than the estimated GC% of coding DNA of 43%. These results suggest that the GC richness of transposable elements, especially that of LINEs, primarily accounted for the greater abundance of TEs in the Bam HI BESs.
The construction of a complete physical map is a vital task of genome sequencing projects. BESs are useful for identifying minimally overlapping clones that extend in each direction from finished clones. Unique paired-end clones are particularly useful for validating, ordering, and joining contigs. Therefore, BACs and their end sequences can be effectively used for integration of linkage and physical maps [12, 28, 29]. However, the possibility of mismapping, mainly due to sequence contamination must be considered. A BAC-based physical map can suffer from chimeric clones, genome assembly errors, and repetitive elements in the genome . To reduce the incidence of incorrect mapping, tools such as repeat-masked BESs and BLAST searching with stringent criteria are necessary. In addition, DNA markers are helpful to detect incorrectly mapped clones. Contigs with two markers from different linkage groups should be tested for clone contamination . Incorrect mapping can also be detectable as an inconsistency in the physical map when a deep coverage BAC library is used. This BLAST-based analysis revealed that the majority of BESs had BLAST hits with ≥ 99% identity against two available WGS data sets. Moreover, the percent identity of BLAST hits against BGI data tended to be slightly lower than that against SGP data, although the main cause of this tendency could not be determined by our analysis. The estimated sequence divergence between the p50T and p50 strains was too low to determine whether the divergence was polymorphism-derived. Therefore, merging of the two WGS data sets is reasonable and will contribute to the construction of a more useful genomic resource in the future.
Characterization of BESs from two BAC libraries confirmed that BAC libraries by nature tend to have certain biases. Therefore, BESs from multiple complementary BAC libraries constructed with different restriction enzymes are a more useful genomic resource. The BESs produced by this research constitute a valuable resource for genomic research in Bombyx mori, for example, as a base for construction of a BAC-based physical map and for exploration of DNA makers. The GenBank accession numbers of the obtained end sequences are DE283657–DE378560.
We used the inbred silkworm strain p50T for the research.
We used two silkworm BAC libraries for end-sequencing, One library was constructed from a partial Eco RI (EC 22.214.171.124) digest of genomic DNA. The construction of this library was reported previously . Copies are available through BACPAC RESOURCES at the Children's Hospital Oakland Research Institute . The other library, prepared by using Bam HI as the restriction enzyme (EC 126.96.36.199), was purchased from the Laboratory for Plant Genomics and GENEfinder Genomic Resource of Texas A&M University . The properties of the two BAC libraries are summarized in Table 1.
Purification of BAC clones
Escherichia coli cells harboring single BAC clones were maintained at -80°C. A fresh colony from each clone was inoculated into each well of a 96-deep-well plate filled with 1.25 mL of 2× LB medium (2% tryptone peptone, 1% yeast extract, and 1% sodium chloride) containing 20 μg/ml chloramphenicol. They were cultured with shaking for 18 to 20 h at 37°C. BAC DNA was prepared using an automated DNA isolation system (PI-1100, Kurabo Industries Ltd., Osaka, Japan) according to the manufacturer's instructions.
Sequencing of BAC ends
Sequencing reactions were performed with 3 μL Big Dye terminator mix (Applied Biosystems, Foster City, CA, USA), 1.0 μL 5× sequencing buffer, 0.5 to 1.0 μg template DNA, 10 pmol of primer, and 4 mM MgCl2. The conditions for the thermal cycling reactions were 96°C for 5 min, then 99 cycles of 96°C for 30 s, 55°C for 10 s and 60°C for 4 min, followed by holding at 4°C. We used custom T7 and SP6 sequencing primers. The DNA was recovered by using MultiScreen 384SEQ plates (Millipore, Billerica, MA, USA).
Base-calling and trimming of BESs were performed with RAMEN, which was used for vector-trimming of silkworm WGS sequences . A BLAST search of mtDNA sequences among the BESs was performed to identify and discard contaminated sequences (e-value: 1e-50). The obtained BESs have been deposited in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank under accession numbers DE283657 to DE378560.
BES clustering was done with the in-house program "Combined BLAST and PhredPhrap" (CBP), which was developed mainly for clustering silkworm ESTs. This program internally uses BLAST  and PHRAP [50, 51]. To optimize the clustering of the BESs, we modified the algorithm slightly. An outline of the clustering procedure follows.
Step 1 An all-to-all BLAST (BLASTN) operation of the BESs was performed. The expectation value (-e option) was set to 10, and no complexity filter (-F option) was used. The number of alignments to be reported (-b option) and maximum number of sequence bases to be created in a volume (-v option) were set to 1000000.
Step 2 Each BLAST hit was analyzed. A provisional cluster was created when a BLAST hit had an identity of at least 90% (Tpid) and an alignment length of 90 bp (Taln). The longest sequence in each provisional cluster was chosen as the representative sequence. A provisional cluster of size 1 was treated as a "singleton."
Step 3 Sequences in each provisional cluster were assembled with PHRAP (using default parameters).
Step4 Reclustering and reassembling were performed under more stringent conditions if multiple contigs were generated. This process was iterated until a single contig was generated. For each iteration, the criterion of alignment length Taln was incremented by 30 bp if Taln was less than or equal to 300 bp. If Taln was greater than 300 bp, the incrementation of Taln was set to 15 bp. If a single contig was not generated by these iterations, then this process was iterated with a stricter Tpid criterion until a single contig was generated. Any unassigned sequences were collected and stored for Step 6.
Step 5 Each contig generated in Step 4 was searched against the member sequences of its own contig for verification. Contigs that did not satisfy the condition, identity ≥ 95% and coverage of alignment ≥ 90%, were stored for Step 6.
Step 6 All sequences stored during the above steps were reprocessed (return to Step 2).
We thank Motoe Sasanuma, Reiko Komatsuzaki, Yoko Fukusaki, Satsuki Tokoro, and Keiko Shiiba for technical assistance. This work was supported by funds from the Ministry of Agriculture, Forestry, and Fisheries of Japan to SS, KM and KY, and from the Bio-oriented Technology Research Advancement Institution to TS and JN.
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