BENCHMARKS
can be derived from genomic fragments that are not true mate pairs. Thus, for scaffolding, we adhere to a conservative policy, using only read pairs in which a junction adapter, the landmark of proper mate-pair structure,
is successfully
identified. To increase the proportion of read pairs harboring the junction adapter, we optimized library insert length and read length. First, to adjust insert length, we performed shearing of circularized DNA with a Covaris S220 sonicator (Covaris, Woburn, MA). We used the same setting as instructed in the standard protocol, except that shearing was repeated from three to seven times, depending on the species, instead of once. The prepared libraries had distinct length distributions, with a considerably higher proportion of DNA molecules shorter than 600 bp, compared with those prepared following the standard protocol (Figure 2). Second, regarding read length, we sequenced the prepared libraries using the Illumina HiSeq rapid run mode with paired-end (PE) 171 cycles with single indexing (2 × 171 + 7 = 349 cycles). This was achieved by using 4 of the TruSeq Rapid SBS v1 kits for 50 cycles, each of which allows no less than 87.5 cycles, including extra cycles for dual indexing, according to the standard protocol in the manual for the SBS kit (4 × 87.5 = 350 cycles). This strategy, employing multiple 50-cycle kits, provides the lowest cost per cycle
Figure 1. Size distributions of tagmented genomic DNA. Genomic DNA was tagmented with the buffer supplied in the kit and with self- made buffer, as well as with variable amounts of enzyme. Electrophoresis was performed using a gel prepared with SeaKem GOLD agarose (LONZA, Basel, Switzerland) in 0.5× TBE on a PippinPulse (Sage Science, Beverly, MA) using its “0.5–15 kb” preset program.
and will also be economical when used with the latest Rapid SBS v2 kit. Overall read properties were analyzed FastQC (
www.bioinformatics.
by
babraham.ac.uk/projects/fastqc/ ), whose recent version (v0.11 or later) reports the proportions of detected Nextera and TruSeq adapters along their base positions in the reads (Supplementary Figure S1). In terms of these measures, an ideal library would exhibit a high proportion of reads in which
the Nextera junction adapter is detected, in addition to a low proportion with the TruSeq adapter. Removal of junction adapters was done using the NextClip (version 1.3) (6) program with default parameters. We compared reads from Library 1, which was prepared according to the standard protocol targeting the mate distance range of 1–6 kb, to those of Library 2, the library prepared according to our modified protocol targeting the same mate distance range
Figure 2. Size distribution of prepared libraries. The red curve shows the length distribution of the library prepared with intensive shearing with Covaris (Library 2), while the blue curve shows the library prepared according to the standard protocol (Library 1). Both libraries were prepared using DNA frag- ments between 1 and 6 kb, size-selected on a BluePippin (Sage Science, Beverly, MA) using the 0.75% gel cassette for 1–10 kb (Catalog # BLF7503) with the built-in program “0.75%DF2–6kb” and measured with a High Sensitivity DNA Kit on Bioanalyzer (Agilent Technologies, Santa Clara, CA).
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