4–6 hr Embryos
+ MiSeq Library Preparation      Individual SeqZip ligation reactions were amplified in individual PCR reactions for 12 cycles using common primers. After amplification, PCR reactions were resolved on a 5% polyacrylamide native gel, and DNA in the size range appropriate for full-length ligation products was cut from the gel and eluted. Eluted DNA was precipitated with isopropanol and amplified for another 22 cycles using primers containing Illumina priming sequences with integrated barcodes. The PCR reactions were purified (Qiagen, QIAquick PCR Purification Kit; 28104) and quantified using a Bioanalyzer 2100 (Agilent) and High Sensitivity DNA Chip. Samples were mixed and submitted for sequencing on the MiSeq instrument using the paired-end, 250 nt read option.    + MiSeq Library Analysis      Dscam1 ligation products are shorter than 400 nt. Therefore, paired-end MiSeq 250 nt reads contain overlapping 3´ sequences. Using these overlapping sequence, paired reads were combined into a single sequence using the Paired-End AssembleR (pear, v. 0.8.1) and default options (Zhang et al. 2014). An index of all possible Dscam1 ligamer combinations was created using single PERL script that permuted all possible ligamer combinations with correct 5´ to 3´ exon 4,6, and 9 ligamer arrangement. ‘Peared’ MiSeq reads were aligned against this index using Bowtie2 v. 2.1.0 (Langmead and Salzberg 2012) in the –very-sensitive-local mode and constrained using –no-discordant to only look for reads where both pairs aligned to the same isoform. Using the SAMtools (v. 0.1.19) software package (H. Li et al. 2009), alignments were furtherer filtered for alignments containing quality 31 and above (-q 31) and read counts per isoform were extracted. Analysis of count values and graph generation was performed using R (R Development Core Team 2008).