NGS Illumina Sequencing
Current project, map the sequence variation(s) underlying the col-4 mutation in N. crassastrain TR1 a his-2; mtr col-4 (single ascospore progeny of FGSC 3017 (a his-2; mtr col-4) and FGSC 2489 (OR74 A). col-4 is genetically defined as being 1 map unit (around 40 kb in this region) right of mtr on LG IV (chr4).
DNA sample (~ 50 ug) sent to Operon for 1 lane of 100 bp paired end sequencing on a HiSeq machine.
QC of DNA sample.
Flash Gel from Operon of the original undigested DNA sample, some degradation, but since the DNA sample will be sheared this did not matter.
QC of DNA sample after size selection of library.
the region around 500 bp was used for library preparation.
Data arrived on an external HD, two ~36 Gb fastq files each containing ~ 145 million 100 bp reads.
My QC of the sequence data done with Fastqc (free from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) installed on my MacBook Air.
Paired end 1
Paired end 2
Really good quality in both cases, interpretation of this figure can be found at the Babraham website (y axis is quality score; higher the better).
Mapping and Analysis of data (so far).
Initial manipulation of the data and the Fastqc analysis was done on a MacBook Air (one file at a time, this is not a laptop really appropriate for such large datasets; 250 Gb hard drive). All the computationally intensive analyses were done on the publicly available Galaxy bioinformatics website (https://main.g2.bx.psu.edu/), but could have been done on command line with the freely available bioinformatics tools mentioned below. Essentially it was mapping the reads to the chr 4 reference sequence of N. crassa from the Broad Institute (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html) with bwa (http://bio-bwa.sourceforge.net/) followed by SNP/indel analysis with the pileup function of samtools (http://samtools.sourceforge.net/).
The original data represents ~600 X coverage of the 40 Mb N. crassa genome. Approx 100 X coverage was taken from this set as sufficient for identification of SNPs and indels (just the first 20 M reads from each paired end file; reads are in the same order in each). The smaller dataset is also more manageable with the above mentioned analysis tools. All analyses below were confirmed with a different set of 20 M reads.
Existing genetic mapping data, and location of the col-4 mutation relative to known genes, helped guide where to look for SNPs/indels, approximately between coordinates 1,827,271 (mtr) and ~1,900,000 (a breakpoint between the reference sequence, in which the col-4 gene is known to reside, and adjacent sequence from a different genetic background, easily discernable from a jump in SNPs/kb). Two candidate SNPs and one indel were identified all within a 4 kb region in a contiguous stretch of approx ~70 kb of reference sequence. No other variation from reference sequence was found. A sample of the raw pileup output (which goes on for ~ 6 M rows or so covering the entire chr 4) is below:
SNP pileup
col 1 - line count from text editor
col 2 -name of the chr4 ref sequence
col 3 - bp coordinates of the ref sequence on chr4
col 4 - reference base call
col 5 - sequence coverage at that base
the rest - individual base calls for each read, with "." meaning reference base, the meaning of some of other characters can be found in the samtools documentation, others, like the alternative bp calls, are obvious.
The main point is a SNP (and N. crassa is haploid) really sticks out. Clearly at position 1,842,866 there is a A to G change in my sequenced strain.
As all three of the variations identified are intergenic, the one above, which is ~100 upstream of a candidate gene is what I will first focus on, actual molecular genetics expts still need to be done to test for the causative variation.
In addition to the above novel variations, the strain sequenced had additional markers that could be confirmed by this sequencing. The strain is mating type a (N. crassa is either A or a), his-2 (histidine auxotroph), and mtr (deficient in the neutral amino acid permease). Mutations in both of these genes were found. For mtr, a C>G change causing a Tyr290 > TAG (stop codon) at ref coordinate 1,826,174 and for his-2 (on chr1, a separate alignment) a G>C at ref coordinate 4,256,930 causing a nonsynonymous Ala > Pro (A32P). Good to have confirmation of known differences.
