Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard W.

doi:10.1371/journal.pone.0055913

Cited by 36 publications

(36 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unlike methylation filtration [28], high-C 0 t fractionation [29] or duplex-specific nuclease digestion [30], complexity reduction by chromosome sorting is lossless and all sequences from a particular chromosome are sequenced. Apart from reducing the complexity of the DNA sample to be sequenced, an important advantage of the targeted approach is that it simplifies DNA sequence analysis by avoiding homoeologues in polyploids (wheat) and paralogues and pseudogenes present on other chromosomes (all species).…”

Section: Discussionmentioning

confidence: 99%

Rapid gene isolation in barley and wheat by mutant chromosome sequencing

et al. 2016

View full text Add to dashboard Cite

Identification of causal mutations in barley and wheat is hampered by their large genomes and suppressed recombination. To overcome these obstacles, we have developed MutChromSeq, a complexity reduction approach based on flow sorting and sequencing of mutant chromosomes, to identify induced mutations by comparison to parental chromosomes. We apply MutChromSeq to six mutants each of the barley Eceriferum-q gene and the wheat Pm2 genes. This approach unambiguously identified single candidate genes that were verified by Sanger sequencing of additional mutants. MutChromSeq enables reference-free forward genetics in barley and wheat, thus opening up their pan-genomes to functional genomics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1082-1) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 99%

Rapid gene isolation in barley and wheat by mutant chromosome sequencing

et al. 2016

View full text Add to dashboard Cite

show abstract

“…() and Matvienko et al. () with modifications. The fragments were then further amplified using Kapa HiFi HotStart ReadyMix and primers (to a final concentration of 0.4 μ m each) to complete the adapters and add a six‐bp index to the P7 adapter (Table S1).…”

Section: Methodsmentioning

confidence: 99%

A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

Owens

Todesco

Drummond

et al. 2017

Molecular Ecology Resources

View full text Add to dashboard Cite

High-throughput sequencing using the Illumina HiSeq platform is a pervasive and critical molecular ecology resource, and has provided the data underlying many recent advances. A recent study has suggested that "index switching," where reads are misattributed to the wrong sample, may be higher in new versions of the HiSeq platform. This has the potential to invalidate both published and in-progress work across the field. Here, we test for evidence of index switching in an exemplar whole-genome shotgun data set sequenced on both the Illumina HiSeq 2500, which should not have the problem, and the Illumina HiSeq X, which may. We leverage unbalanced heterozygotes, which may be produced by index switching, and ask whether the undersequenced allele is more likely to be found in other samples in the same lane than expected based on the allele frequency. Although we validate the sensitivity of this method using simulations, we find that neither the HiSeq 2500 nor the HiSeq X has evidence of index switching. This suggests that, thankfully, index switching may not be a ubiquitous problem in HiSeq X sequence data. Lastly, we provide scripts for applying our method so that index switching can be tested for in other data sets.

show abstract

“…Library normalization was performed using double stranded DNA nuclease (Evrogen) as published before[59]. Four 300-bp libraries from roots, shoots, spike and grain were pooled for normalization.…”

Section: Methodsmentioning

confidence: 99%

Separating homeologs by phasing in the tetraploid wheat transcriptome

et al. 2013

View full text Add to dashboard Cite

BackgroundThe high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs.ResultsA total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing.ConclusionsOur study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.

show abstract

Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

Cited by 36 publications

References 67 publications

Rapid gene isolation in barley and wheat by mutant chromosome sequencing

Rapid gene isolation in barley and wheat by mutant chromosome sequencing

A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

Separating homeologs by phasing in the tetraploid wheat transcriptome

Contact Info

Product

Resources

About