Sequence map of the 3-Mb Giardia duodenalis assemblage A chromosome

Abstract

The genome of the gut protozoan parasite Giardia duodenalis (assemblage A) has been sequenced and compiled as contigs and scaffolds (GiardiaDB- http://GiardiaDB.org ), but specific chromosome location of all scaffolds is unknown. To determine which scaffolds belong to the 3-Mb chromosome, a library of probes specific for this chromosome was constructed. The probes were hybridised to NotI-cleaved whole chromosomes, and the combined size of different NotI segments identified by the probes was 2,225 kb indicating the probes were well distributed along the 3-Mb chromosome. Six scaffolds (CH991814, CH991779, CH991793, CH991763, CH991764, and CH991761) were identified as belonging to the 3-Mb chromosome, and these scaffolds were ordered and oriented according to scaffold features including I-PpoI sites and hybridisation pattern. However, the combined size of scaffolds was more than 4 Mb. Approximately, 1 Mb of scaffold CH991763 carrying previously identified sequences specific for the 1.5-Mb chromosome(s) including subtelomeric sequence was reassigned, and several other anomalies were addressed such that the final size of the apparently 3-Mb chromosome is estimated to be 2,885 kb. This work addresses erroneous computer-based assignment of a number of contigs and emphasises the need for alternative and confirmatory methods of scaffold construction.

Chromosome sequence maps of the Giardia lamblia assemblage A isolate WB

“…In G. lamblia it also cleaves the LSrRNA gene once, and as previously reported [9,12,16], is a useful tool to remove rRNA gene tandem arrays located at the downstream end of each chromosome. The removal of the arrays allows chromosomes to more readily enter agarose gels leading to improved chromosome separation [12]. However, I- Ppo I also cleaves the 3 Mb chromosome 4, and this was a useful mapping tool [12].…”

“…It has a 13 to15 bp recognition sequence which appears to be conserved in the LSrRNA genes of higher eukaryotes [34]. In G. lamblia it also cleaves the LSrRNA gene once, and as previously reported [9,12,16], is a useful tool to remove rRNA gene tandem arrays located at the downstream end of each chromosome. The removal of the arrays allows chromosomes to more readily enter agarose gels leading to improved chromosome separation [12].…”

“…Nonetheless, the advent of pulsed field gel electrophoresis (PFGE) for separating large DNA molecules up to 5 Mb (Figure 1) [10] and rapid large scale sequencing techniques [11] have allowed a better understanding of the Giardia genome. What has been missing until recently was the integration of PFGE data and the genome sequence [12,13]. This review addresses how whole chromosome separation and publically available Giardia genome sequence data were used to construct a working map of the chromosomes of G. lamblia assemblage A isolate WB.…”

“…Such markers were used, initially, to map 13 Not I segments (totaling 1.8 Mb) of the 2 Mb chromosome [31]. When the genome sequence of WBC6 (a cloned line of the WB isolate) was released and the Giardia genome database (GiardiaDB) was made available at http://www.giardiadb.org [32], the 2 Mb chromosome map, with the help of one representative probe sequence from each Not I segment [13,31] (GenBank accession numbers L49329–L49335) was confirmed, and this inspired further mapping [12]. The 2 Mb and the other chromosomes have been variously numbered in the literature (Box 2).…”

“…The 2 Mb and the other chromosomes have been variously numbered in the literature (Box 2). Compilation of the chromosome sequence maps has used other resources in conjunction with GiardiaDB and the role of three key restriction enzymes, Xba I, I- Ppo I and Not I, and a collection of chromosome-specific probes [9,12,13,22,33] in mapping G. lamblia isolate WB chromosomes are described below. The strategy used to map the chromosomes is outlined in Box 3.…”