Instagress = Instantaneous introgression
Nine DNA samples with unique IDs FRAG01448-FRAG01456 were processed.
PCR-libraries were made using KAPA Hyper kit and 150PE sequencing performed on HiSeq4000 by QB3.
- Alignment of reads using bwa
Raw reads were interleaved, filtered and shortened to 50bp.
Aligned by using bwa-do-all on a database that has the TAIR10 reference genome as well as the T-DNA insert sequence from pCAMBIA.
*Pairs were mapped as single reads for this analysis. This is important because bwa's paired-end function with salvage does not consider mis-mapping due to 'Far Pairs' and in our experience, and using longer reads that span unique junctions prevent proper mapping resulting in fewer informative reads (see data below).
bwa-doall-vModules-current.py -d ~ekhtan/INSTAGRESS/genomes/combined_pCAMBIA_TAIR10.fa -O -t 16
Hits to pCAMBIA based on mapping using 50bp vs 150bp is shown here.
Hits to pCAMBIA using 50bp: grep -c pCAM *.sam
FRAG01448_aln.sam:174
FRAG01449_aln.sam:28
FRAG01450_aln.sam:106
FRAG01451_aln.sam:1771
FRAG01452_aln.sam:593
FRAG01453_aln.sam:1170
FRAG01454_aln.sam:315
FRAG01455_aln.sam:578
FRAG01456_aln.sam:227
Hits to pCAMBIA using 150bp: grep -c pCAM *.sam
FRAG01448_aln.sam:147
FRAG01449_aln.sam:24
FRAG01450_aln.sam:99
FRAG01451_aln.sam:1722
FRAG01452_aln.sam:522
FRAG01453_aln.sam:1127
FRAG01454_aln.sam:289
FRAG01455_aln.sam:520
FRAG01456_aln.sam:212
###FarPairs Analysis
This is a cursory look at the unique breakpoint junctions for T-DNA insertion sites. This step facilitates the de novo assembly of reads from target regions.
for i in *.sam
do
SegregatePE.py $i 5000
done
This is an example output from the script for FRAG01451:
Total:
8278497
TruePairs:
4915803
Average distance of TruePairs:
284.656289929
FarPairs:
80677
OddPairs:
415329
BadPairs:
2866688
Unpaired:
0
CheckTotal:
8278497
Because we know that the cut sites were on Chr2 this is an example on how this data is useful:
grep pCAMBIA *_FarPairs.txt | grep Chr2
K00188:47:H3NV2BBXX:3:1108:9211:41088 16 Chr2 75666 16 pCAMBIA_fwa 73 No NA TCGCTGGGGCCTCCGCCCACATCTACTCTTCCCAGCCCTTCCACGATGTC GTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGG
K00188:47:H3NV2BBXX:3:1108:6025:48896 0 Chr2 75278 16 pCAMBIA_fwa 220 No NA GTGTTACAGACTAGTTTACTGAGTCCGGTGGGTTACAGGGCGGGCGGCGA CCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTA
K00188:47:H3NV2BBXX:3:1111:29061:14361 16 pCAMBIA_fwa 90 16 Chr2 75459 No NA AATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAAT ATTCTAAGGCCCAAACCATTATATTAAGTTGGTTGGTCCACCAAAGTCAG
K00188:47:H3NV2BBXX:3:1113:28726:10229 16 pCAMBIA_fwa 69 0 Chr2 75216 No NA TAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTA GCCCCTCCATTTTCGGAACTTGCTCTTCTCCAAGCTAAATCTTATC
K00188:47:H3NV2BBXX:3:1123:20851:6501 16 pCAMBIA_fwa 63 16 Chr2 75627 No NA CGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATT CGCTGCTGTGCCCATTTCTTCTATCATTACACTTTAGAATCGCTGGGGCC
K00188:47:H3NV2BBXX:3:1126:19116:46328 0 Chr2 75259 16 pCAMBIA_fwa 235 No NA ATCTGAAATAGAAGATATTGTGTTACAGACTAGTTTACTGAGTCCGGTGG AATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGT
K00188:47:H3NV2BBXX:3:1206:8136:27408 0 Chr2 75180 16 pCAMBIA_fwa 95 No NA ATCTACGAACCTGTGGGTGGTGGATCTATACACGACGCCCCTCCATTTTC ATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAAT
K00188:47:H3NV2BBXX:3:1208:2767:23557 16 pCAMBIA_fwa 224 16 Chr2 75450 No NA ATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGA GCCCAAACCATTCTAAGGCCCAAACCATTATATTAAGTTGGTTGGTCCAC
- Finding instagressed T-DNA sites
Taking the LB and RB of the T-DNA insert, we will start assembling breakpoint junctions from the fq reads from 500bp from the LB and RB of the pCAMBIA T-DNA sequence.
Make a bins_pCAMBIA.txt file that look like this:
Chrom BinStart BinEnd
pCAMBIA_fwa 0 500
pCAMBIA_fwa 6368 6868
Run binsearch algorithm. Note that the full 150bp PE fq files are used in conjunction with sam files that were mapped based on 50bp reads.
batch-specific-junction-bin-search.py -b bins_pCAMBIA.txt -Q
Perform PRICE assembly on these extracted reads, followed by BLASTn.
batch-uninterleaver-PRICE-Blaster.py ~ekhtan/INSTAGRESS/genomes/combined_pCAMBIA_TAIR10.blastn
Example of Blastn ouput that shows the potential integration sites, which matches the original T-DNA integration sites
# BLASTN 2.2.31+
# Query: contig_2 (841nt) unchanged
# Database: /home/ekhtan/INSTAGRESS/genomes/combined_pCAMBIA_TAIR10.blastn
# Fields: query id, subject id, % identity, alignment length, mismatches, gap opens, q. start, q. end, s. start, s. end, evalue, bit score
# 3 hits found
contig_2 pCAMBIA_fwa 100.00 422 0 0 420 841 6597 6176 0.0 780
contig_2 pCAMBIA_fwa 99.13 231 2 0 349 579 47 277 4e-115 416
contig_2 Chr2 100.00 324 0 0 1 324 75715 75392 3e-170 599
# BLASTN 2.2.31+
# Query: contig_3 (621nt) unchanged
# Database: /home/ekhtan/INSTAGRESS/genomes/combined_pCAMBIA_TAIR10.blastn
# Fields: query id, subject id, % identity, alignment length, mismatches, gap opens, q. start, q. end, s. start, s. end, evalue, bit score
# 3 hits found
contig_3 pCAMBIA_fwa 100.00 488 0 0 134 621 21 508 0.0 902
contig_3 pCAMBIA_fwa 98.75 160 2 0 231 390 6597 6438 8e-76 285
contig_3 Chr2 100.00 119 0 0 1 119 75259 75377 2e-56 220
Original insertion site is at Chr2:752,377 to LB and Chr2:752,392 at to RB of pCAMBIA from what I can tell.
If real, that means the introgression is via the HR pathway/gene conversion (?), and not through NHEJ-associated cut and paste.
Two out of the nine lines sequenced (FRAG01451 - plant #12 and FRAG01453 - plant #18) have junction support for an instagress-event on the targeted site on Chr2.
- Caveats
The sgRNA cut sites also contain ~100bp of Chr2 as part of 'donor' pCAMBIA T-DNA from GFP-tailswap parent. Therefore, reconstruction using 150 bp PE reads and PRICE assembly did not yield a clear picture although the majority shows the event as described above.
Solution: PCR oligos 500bp outside the PAM sites for Sanger sequencing has been ordered.