cells can be extremely tedious. The use of multiple long PCR reactions or RCA is also intimidating with the gaps due to PCR failure. An
innovative method was able to sequence total
DNA including nuclear, mitochondrial, and
chloroplast genome by skipping any experimental isolation of pure cpDNA (Nock et al. 2011).
The separation occurred in the next step by using
computational filtration to remove any contaminated DNA derived from nucleus and mitochondria. Three duckweed chloroplast genomes
(Spirodela polyrhiza, Wolffiella lingulata, and
Wolffia australiana) were sequenced by using
such method and were assembled without any
experimental purification (Table 10.2). The data
have shown that the reads of total DNA
sequencing from a quadrant slide without any
purification could reach more than 1000-fold
coverage (Wang and Messing 2011).
10.3 Chloroplast Genome
Sequencing and Annotation
It is well known that a chloroplast genome size is
relatively small (*107 to 218 Kb) (Daniell et al.
2016). After removing one copy of inverted
repeat, the size becomes comparable to a BAC
clone. The chloroplast genome is generally a
by-product of a whole nuclear genome
sequencing project that could be obtained from a
fosmid or a BAC clone under Sanger sequencing
platform (Sanger 1988). With the rapid development of high-throughput next-generation
sequencing (NGS) (Schuster 2008), it substantially facilitates the releases of chloroplast genomes. However, the short reads (*100 bp) from
NGS are GC biased and cannot fully span over
the junctions of inverted repeats (IRs), resulting
in incomplete genome and unsolved IRs. PacBio
reads become promising to overcome the
assembly challenge due to the long reads
(*10 Kb) that can specifically determine the
sequence location in the genome (Fig. 10.2) (Eid
et al. 2009).
10.3.1 Chloroplast Genome of L.
minor Sequenced
by Sanger Technology
The chloroplast genome of L. minor (common
duckweeds) was sequenced by Sanger sequencer
in 2008 (Mardanov et al. 2008). The cpDNA was
prepared by the amplified PCR fragments ranging from 1 to 8 Kb. Each fragment was automatically sequenced on ABI sequencers using
the BigDye Terminator. All fragments were
sequenced *6 times on average with the way of
primer walking. Assembling was performed with
the Gene Studio program (http://www.
genestudio.com). L. minor chloroplast genome
had a size of 165,955 bp in a circular molecule,
including a pair of 31,223-bp inverted repeat
regions, an 89,906-bp large single copy, and a
13,603-bp small single copy (Table 10.2). L.
minor had a tendency of expansion in terms of
the inverted repeats in comparison with other
monocots. The genes of infA, ycf15, and ycf68
were absent from L. minor, but present in other
plant chloroplast genomes. The tRNA types
found in L. minor chloroplast genome that could
recognize all plastid codons (Mardanov et al.
2008).
10.3.2 Chloroplast Genomes of S.
polyrhiza, W. lingulata
and W. australiana
Sequenced by NGS
Technology
The number of sequenced plant chloroplast genomes has exploded to more than 1000 species
mostly due to the availability of NGS at cheaper
sequencing cost (Jansen et al. 2005). A multiplex
sequencing-by-synthesis approach using the Illumina Genome Analyzer sequenced the chloroplast
genomes of Picea sitchensis and seven pine species simultaneously. The pooled PCR-amplified
products were ligated and multiplexed to the
adapters including 3-bp indexing tags. The
10 Duckweed Chloroplast Genome Sequencing and Annotation
109
innovative method was able to sequence total
DNA including nuclear, mitochondrial, and
chloroplast genome by skipping any experimental isolation of pure cpDNA (Nock et al. 2011).
The separation occurred in the next step by using
computational filtration to remove any contaminated DNA derived from nucleus and mitochondria. Three duckweed chloroplast genomes
(Spirodela polyrhiza, Wolffiella lingulata, and
Wolffia australiana) were sequenced by using
such method and were assembled without any
experimental purification (Table 10.2). The data
have shown that the reads of total DNA
sequencing from a quadrant slide without any
purification could reach more than 1000-fold
coverage (Wang and Messing 2011).
10.3 Chloroplast Genome
Sequencing and Annotation
It is well known that a chloroplast genome size is
relatively small (*107 to 218 Kb) (Daniell et al.
2016). After removing one copy of inverted
repeat, the size becomes comparable to a BAC
clone. The chloroplast genome is generally a
by-product of a whole nuclear genome
sequencing project that could be obtained from a
fosmid or a BAC clone under Sanger sequencing
platform (Sanger 1988). With the rapid development of high-throughput next-generation
sequencing (NGS) (Schuster 2008), it substantially facilitates the releases of chloroplast genomes. However, the short reads (*100 bp) from
NGS are GC biased and cannot fully span over
the junctions of inverted repeats (IRs), resulting
in incomplete genome and unsolved IRs. PacBio
reads become promising to overcome the
assembly challenge due to the long reads
(*10 Kb) that can specifically determine the
sequence location in the genome (Fig. 10.2) (Eid
et al. 2009).
10.3.1 Chloroplast Genome of L.
minor Sequenced
by Sanger Technology
The chloroplast genome of L. minor (common
duckweeds) was sequenced by Sanger sequencer
in 2008 (Mardanov et al. 2008). The cpDNA was
prepared by the amplified PCR fragments ranging from 1 to 8 Kb. Each fragment was automatically sequenced on ABI sequencers using
the BigDye Terminator. All fragments were
sequenced *6 times on average with the way of
primer walking. Assembling was performed with
the Gene Studio program (http://www.
genestudio.com). L. minor chloroplast genome
had a size of 165,955 bp in a circular molecule,
including a pair of 31,223-bp inverted repeat
regions, an 89,906-bp large single copy, and a
13,603-bp small single copy (Table 10.2). L.
minor had a tendency of expansion in terms of
the inverted repeats in comparison with other
monocots. The genes of infA, ycf15, and ycf68
were absent from L. minor, but present in other
plant chloroplast genomes. The tRNA types
found in L. minor chloroplast genome that could
recognize all plastid codons (Mardanov et al.
2008).
10.3.2 Chloroplast Genomes of S.
polyrhiza, W. lingulata
and W. australiana
Sequenced by NGS
Technology
The number of sequenced plant chloroplast genomes has exploded to more than 1000 species
mostly due to the availability of NGS at cheaper
sequencing cost (Jansen et al. 2005). A multiplex
sequencing-by-synthesis approach using the Illumina Genome Analyzer sequenced the chloroplast
genomes of Picea sitchensis and seven pine species simultaneously. The pooled PCR-amplified
products were ligated and multiplexed to the
adapters including 3-bp indexing tags. The
10 Duckweed Chloroplast Genome Sequencing and Annotation
109