The program KmerGenie v.1.5924 [ 79] was used to calculate the best k-mer value for each assembly from k = 23 to the maximal possible k = 223 in the default two passes.
On the basis of these results, we used a k-mer value of 61 for de novo assembly of alfalfa EST reads.
We calculated the fitted relative k-mer values for the sex chromosome analysis as follows: cr′ i, j, m = f j, m (GC i, j ) · (j = X, Y), for the fitted relative k-mer coverage from a regression of an adult male data set; and cr′ i, j, f = f j, f (GC i, j ) · (j = X, Y), for the fitted relative k-mer coverage from a regression of a fetal-female data set.
We used K-mer values of 29 for the six samples with single reads and 31 for the remaining samples with paired-end reads.
In case of ABySS and Velvet assembly, multiple k-mer approach with every other k-mer values from 21 to 95 for ABySS and from 45 to 95 for Velvet were used so as to maximize assembly contiguity and sensitivity.
The VelvetOptimiser script (version 2.2.4) was run for all odd k-mer values from 21 to 99.
If one contig were assembled with each k-mer value (2-63) and for 0-3 mismathens, then 20x4 (80) full length identical transcripts are expected for each gene.
A k-mer value was initially set for each accession according to a linear model 'K=2*int (0.38*(sequencing depth) +10)+1', which was trained from 50 randomly selected rice accessions.
The k-mer value was set to 31 for velveth, and the coverage cut-off and expected coverage was set to 'auto' in velvetg.
The de novo assembly of the clean reads was carried out using the SOAPdenovo program (http://soap.genomics.org.cn/soapdenovo.html) with the default settings, except for the k-mer value, which was set at a specific chosen value [ 32].
De novo assembly of the clean reads was performed using SOAPdenovo http://soap.genomics.org.cn/soapdenovo.html with the default settings, except for the K-mer value, which was set at a specific value [ 42].
