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Table 1. Properties of the prepared mate-pair libraries. Peak in library
Library Targeted mate distance in size selection (kb)
Library 1
Library 2 Library 3
1–6 1–6 11–18
size distribution* (bp)
869
545 501
# of PCR cycles
10 8 10
Read length (nt)**
101 171 171 171
Duplicate read pairs (%)
0.48 0.38 0.60 3.08
Proportion of usable read pairs*** (%)
33.6 59.9 65.4 68.1
Note that for each library 15 million read pairs were randomly sampled with the program seqtk (
https://github.com/lh3/seqtk) and used in the measures of duplicate read pair % and the proportions of usable reads by the program NextClip.
*This column shows the values reported by the Bioanalyzer (see Supplementary Figure S1).
**Reads with a length of 101 nucleotides (nt) were obtained by trimming the end of 171 nt reads using the program fastx_trimmer (
http://hannonlab.cshl.edu/fastx_toolkit/).
***This column shows the proportion of read pairs that have a junction adaptor and are longer than 25 nt after the removal of the adaptor.
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(Table 1). The following statistics are based on random sampling of an adjusted number of sequenced reads among the different libraries (15 million read pairs). In comparison to Library 1, sequencing Library 2 with PE 171 cycles resulted in the largest proportion of read pairs (65.4%) that are regarded as usable in genome scaffolding (those in Categories A, B and C as defined by NextClip) (Table 1). This comparison shows that longer reads largely contribute to the increased proportion of usable reads, an effect that is enhanced with intensive shearing. Moreover, we sequenced a library prepared according to our modified protocol targeting the mate distance range of 11–18 kb (Library 3), and this library yielded a comparable proportion of usable reads with only 10 PCR cycles (Table 1). Using the mate-pair reads, we
computed tentative genome scaffolding using the Platanus (version 1.2.1) program (7). Scaffolding with mate-pair reads extensively increased the overall sequence continuity, as shown by the increased N50 values. Moreover, these
libraries provided improved coverage of the genome (Table 2) as indicated by the proportion of core eukaryotic genes (CEG) detected with the program CEGMA version 2.4 (8). Scaffolding using reads from Library 2 sequenced with 171 cycles had the best score (Table 2), albeit evaluation of genome assemblies requires multifaceted assessment (9). The power of our modified protocol was most evident when multiple libraries with varied mate distances were employed in scaffolding (Table 2). In our trials using P. picta genomic
DNA, we targeted relatively broad size ranges of tagmented DNA fragments (1–6 kb and 11–18 kb), but for a different species, we succeeded in preparing mate-pair libraries with narrower mate distance ranges (e.g., 12–16 kb). It is of note that our protocol enabled library preparation with no more than 10 PCR cycles even for >10 kb mate distance to yield 20 l of library solution of up to 7 nM (0.14 pmol), which kept the PCR duplicate rates down to less than 4% among 15 million read pairs (Table 1).
Table 2. Contribution of mate-pair reads to genome scaffolding. Reads used* PE only
PE + MP (Library 1, 101 nt) PE + MP (Library 1, 171 nt) PE + MP (Library 2, 171 nt)
PE + MP (Libraries 2 and 3, both 171 nt)
Scaffold N50 (kb) Max scaffold length (kb) Gene coverage (%)** 13.3 25.9 41.0 51.8
382.9 1819.1
1420.0 1790.6 1792.6 2425.0
58.9 71.3 75.4 77.4 82.7
*This column indicates read types [paired-end (PE) or mate-pair (MP)] and choice of MP libraries used in the scaffolding step after assembling only paired-end (PE) reads.
**This column shows the numbers of core eukaryotic genes (CEGs) detected as “complete” by CEGMA runs with the “--vrt” option.
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