depth is deep enough and reads are long, each homolog/haplotype can be separated
prior to assembly.
3.25 mRNA/cDNA/EST/RNA-Seq Data
The discovery and proper annotation of genes is greatly facilitated by utilization of
sequence obtained from mRNA molecules. Since RNA is difficult to sequence, all
first- and second-generation sequencing techniques have researchers convert mRNA
into complementary DNA (cDNA) using reverse transcriptase.
11 Partial cDNA
sequences have been referred to as expressed sequence tags (ESTs), while highthroughput sequencing of cDNA using second-generation techniques is called
RNA-Seq. Regardless, cDNA sequences (particularly relatively long cDNA
sequences) greatly facilitate structural genome annotation (see below). RNA-Seq
data can also be useful, although it is perhaps best utilized to study differential gene
expression (especially after a quality genome sequence is in place).
3.26 Proteogenomic Mapping
cDNA sequencing can help one learn which genes in a genome are being transcribed
to create mRNA. However, not all RNA sequences that are transcribed are translated
into protein. Proteogenomic mapping is the use of proteomics (mass spectroscopy)
data as a means to facilitate recognition of genes that are actually being translated
(Jaffe et al. 2004).
3.27 Structural Annotation
Structural annotation is the process of identifying and demarcating certain sequence
types within DNA (or RNA or protein) sequence data. Typically, structural
annotation is performed using algorithms that recognize specific motifs (ab initio
identification) and/or compare known sequence types to the query sequence
(homology-based identification). Unless noted otherwise, the features mentioned
below can be identified using both ab initio and homology-based methods:
• Open reading frames (ORFs) – An ORF is any stretch of DNA that has a
transcription start codon (5
0 -ATG) and a transcription stop codon (5
0 -TAA,
TGA, or TAG) separated by a distance that suggests a potential coding region
11 Oxford Nanopore and PacBio have started advertising direct RNA sequencing.
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145
prior to assembly.
3.25 mRNA/cDNA/EST/RNA-Seq Data
The discovery and proper annotation of genes is greatly facilitated by utilization of
sequence obtained from mRNA molecules. Since RNA is difficult to sequence, all
first- and second-generation sequencing techniques have researchers convert mRNA
into complementary DNA (cDNA) using reverse transcriptase.
11 Partial cDNA
sequences have been referred to as expressed sequence tags (ESTs), while highthroughput sequencing of cDNA using second-generation techniques is called
RNA-Seq. Regardless, cDNA sequences (particularly relatively long cDNA
sequences) greatly facilitate structural genome annotation (see below). RNA-Seq
data can also be useful, although it is perhaps best utilized to study differential gene
expression (especially after a quality genome sequence is in place).
3.26 Proteogenomic Mapping
cDNA sequencing can help one learn which genes in a genome are being transcribed
to create mRNA. However, not all RNA sequences that are transcribed are translated
into protein. Proteogenomic mapping is the use of proteomics (mass spectroscopy)
data as a means to facilitate recognition of genes that are actually being translated
(Jaffe et al. 2004).
3.27 Structural Annotation
Structural annotation is the process of identifying and demarcating certain sequence
types within DNA (or RNA or protein) sequence data. Typically, structural
annotation is performed using algorithms that recognize specific motifs (ab initio
identification) and/or compare known sequence types to the query sequence
(homology-based identification). Unless noted otherwise, the features mentioned
below can be identified using both ab initio and homology-based methods:
• Open reading frames (ORFs) – An ORF is any stretch of DNA that has a
transcription start codon (5
0 -ATG) and a transcription stop codon (5
0 -TAA,
TGA, or TAG) separated by a distance that suggests a potential coding region
11 Oxford Nanopore and PacBio have started advertising direct RNA sequencing.
Sequencing Plant Genomes
145