DNA Sequence-Based „Bar Codes” for Tracking the Origins of Expressed Sequence Tags from a Maize cDNA Library Constructed Using Multiple mRNA Sources

Abstract

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.

Nearly Identical Paralogs: Implications for Maize (Zea mays L.) Genome Evolution

“…These were then subjected to touchdown RT-PCR using the pooled inbred line B73 cDNA, very similar to that described previously (Fu et al 2005). In addition, RNA samples were also isolated from various tissues, organs, and developmental stages of the B73 inbred line similar to those described by Qiu et al (2003). Reactions that yielded single bands that were not larger than the genomic PCR product were sequenced.…”

Tunable Genotyping-By-Sequencing (tGBS®) Enables Reliable Genotyping of Heterozygous Loci

“…Comparison of tGBS with conventional GBS To explore the advantages of tGBS relative to conventional GBS (cGBS), we compared tGBS data generated from the NAM founders presented in this paper with cGBS data generated from a large diversity panel by Romay et al (2013) (Romay et al 2013). Because different samples were genotyped using tGBS and cGBS, it was not possible to directly compare the SNP genotypes generated by the two technologies.…”

“…In contrast to other methods (van Orsouw et al 2007;Andolfatto et al 2011;Elshire et al 2011;Peterson et al 2012;Wang et al 2012;Stolle and Moritz 2013) which employ double-stranded adapters, a single-strand oligonucleotide (oligo) is ligated to each overhang. One of the oligos is unique to an individual sample and contains a DNA barcode (Qiu et al 2003) (barcode oligo) while the other oligo is common to all samples and contains a universal sequence (universal oligo) for subsequent library construction. Following ligation, two PCR steps complete the construction of the sequencing library.…”

“…Alignment of reads to reference genome Cleaned reads were aligned to the B73 reference genome (AGP v2) (Schnable et al 2009) using GSNAP (Wu and Nacu 2010). Only confidently mapped reads were used for subsequent analyses, which are uniquely mapped with at least 50 bp aligned, at most 2 mismatches every 40 bp and less than a 3 bp tail for every 100 bp of read.…”

“…Based on their cutting frequencies and abilities to generate appropriate overhangs (one 5' overhang and one 3' overhang), the utility of NspI and BfuCI for tGBS was evaluated by simulation using the maize B73 reference genome (Schnable et al 2009). Constraining the analysis to only non-repetitive DNAfragments with different cut sites on each end with a total size between 100 and 300 bp yielded a total of 246,124, 44,372, and 8,645 non-repetitive DNA fragments for 1-, 2-or 3-base pairs of selective bases (T, TG, and TGT) respectively.…”