Synthetic plasmid Raw sequence reads

We ordered four ultramers from Integrated DNA Technologies: BC1-BC1, BC2-BC2, BC3-BC3 and the adapter (Table \ref{tableoligos}). BC1-BC1, BC2-BC2 and BC3-BC3 contain the Illumina P5-SBS3T sequence followed by a 20nt barcode, the Lambda phage Attachment site L (AttL) sequence and another 20nt barcode. Whereas the barcodes of BC1-BC1 and BC2-BC2 have a balanced base composition, BC3-BC3 has GC rich barcodes with an expected GC content of $80\%$. The adapter oligonucleotide is $5^\prime$ phosphorylated and contains a 15nt barcode followed by the reverse complement of the Illumina P7-SBS8 sequence. It''s $3^\prime$ end is phosphorylated to avoid circularization. We pooled BC1-BC1 and BC2-BC2 in equal proportions and ligated them to the $3^\prime$ adapter using CircLigase ssDNA ligase (epicentre; previously sold as Thermophage ssDNA ligase) as previously described \cite{listructure-independent2006}. For the high GC dataset ZL053 we additionally included BC3-BC3 in the ligation reaction at a fivefold reduced concentration relative to BC1-BC1 and BC2-BC2. The ligation reaction was cleaned up with Agencourt RNAClean XP beads (Beckman Coulter) according to the manufacturers instructions. The ligated products were subjected to 25 cycles of PCR using $47\mu l$ Accuprime Pfx SuperMix (Invitrogen), $1\mu l$ of $10\mu M$ forward and reverse primers each (Table \ref{tableoligos}) and $1\mu l$ input. Cycling was performed in a BioRad MyCycler Thermal Cycler using standard Accuprime protocol with $58^\circ C$ annealing temperature and 30 seconds extension time. The PCR product was gel extracted and sequenced on a single lane of a HiSeq 2000 machine at PE101 per dataset.

我们从整合DNA技术公司（Integrated DNA Technologies）订购了4条超寡核苷酸（ultramers），分别为BC1-BC1、BC2-BC2、BC3-BC3以及衔接子（adapter），具体信息见表
ef{tableoligos}。BC1-BC1、BC2-BC2和BC3-BC3均包含因美纳（Illumina）P5-SBS3T序列，其后依次为一段20 nt条形码（barcode）、λ噬菌体附着位点L（AttL）序列，以及另一段20 nt条形码。BC1-BC1与BC2-BC2的碱基组成均较为均衡，而BC3-BC3的条形码富含GC碱基，预计GC含量为80%。该衔接子寡核苷酸的5′端带有磷酸基团，包含一段15 nt条形码，其后为因美纳P7-SBS8序列的反向互补序列；其3′端亦带有磷酸基团，以避免发生环化。我们将BC1-BC1与BC2-BC2按等比例混合，并使用CircLigase单链DNA连接酶（Epicentre公司；此前以Thermophage单链DNA连接酶的名称销售）进行连接，具体操作参照已发表的方法cite{listructure-independent2006}。针对高GC数据集ZL053，我们在连接反应中额外加入了BC3-BC3，其浓度相较于BC1-BC1与BC2-BC2降低了五倍。我们按照制造商的操作说明，使用Agencourt RNAClean XP磁珠（贝克曼库尔特公司）对连接反应体系进行纯化。将连接产物置于体系中进行25个循环的PCR扩增，体系包含47 μL Accuprime Pfx预混酶（Invitrogen公司）、各1 μL浓度为10 μM的正向与反向引物（见表
ef{tableoligos}）以及1 μL模板。PCR扩增在伯乐（BioRad）MyCycler热循环仪中进行，采用标准Accuprime反应程序：退火温度为58℃，延伸时长为30秒。PCR产物经胶回收后，在HiSeq 2000测序仪的单个泳道上进行双端101 bp测序，每个数据集均采用此流程。