Methodology Language for Arachis CP#3906 & #3899 for assembly


Two Chicago libraries were prepared as described previously (Putnam et al, 2016). Briefly, for each
library, ~500ng of HMW gDNA (mean fragment length = 90kb) was reconstituted into chromatin in vitro
and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5’ overhangs filled in with
biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were
reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not
internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing
libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing
fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries
were sequenced externally.

The input de novo assembly, shotgun reads, and Chicago library reads were used as input data for Mecat
and HiRise, a software pipeline designed specifically for using proximity ligation data to scaffold
genome assemblies (Putnam et al, 2016). Shotgun and Chicago library sequences were aligned to the draft
input assembly using a modified SNAP read mapper (http://snap.cs.berkeley.edu). The separations of
Chicago read pairs mapped within draft scaffolds were analyzed by HiRise to produce a likelihood model
for genomic distance between read pairs, and the model was used to identify and break putative misjoins,
to score prospective joins, and make joins above a threshold. After scaffolding, shotgun sequences
were used to close gaps between contigs.


References: 1. Putnam NH, O'Connell BL, Stites JC, Rice BJ, Blanchette M, Calef R, Troll CJ, Fields
A, Hartley PD, Sugnet CW, Haussler D, Rokhsar DS, Green RE. Genome Research. 2016 Mar;26(3):342-50.
2. Erez Lieberman-Aiden, Nynke L. van Berkum,Louise Williams, Maxim Imakaev, Tobias Ragoczy, Agnes
Telling, Ido Amit, Bryan R. Lajoie, Peter J. Sabo, Michael O. Dorschner, Richard Sandstrom, Bradley
Bernstein, M. A. Bender, Mark Groudine, Andreas Gnirke, John Stamatoyannopoulos, Leonid A. Mirny, Eric
S. Lander, Job Dekker. Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of
the Human Genome. Science. 2009; 326, 289.  3. Chapman JA, Ho I, Sunkara S, Luo S, Schroth GP, Rokhsar
DS. Meraculous: de novo genome assembly with short paired-end reads. PLoS One. 2011;6(8):e23501.  4.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics.
2014 Aug 1;30(15):2114-20.


*N50 is defined as the scaffold length such that the sum of the lengths of all scaffolds of this size
or less is equal to 50% of the total assembly length