Are these little contigs just rare assembled haplotypes? Check for low input read mapping rates and use for blobtools
Map long reads
/work/gif/remkv6/Toth/12_Bombus_dahlbomii/14_Blobtools
ln -s ../10_FindBuscos/30_537121167.hifi_reads.fastq.gz
ln -s ../13_ReAnnotate/ConcatDahlbomiiGenome.FINAL.fasta
echo "sh runMinimapNbamSort.sh 30_537121167.hifi_reads.fastq.gz ConcatDahlbomiiGenome.FINAL.fasta" >ReadMapping.sh
#runMinimap.sh
##############################################################################
#!/bin/bash
query=$1
target=$2
outname="${query%.*}_${target%.*}_minimap2.sam"
module load minimap2
minimap2 -x asm5 -a -t 36 $target $query > ${outname}
ml samtools;samtools view --threads 36 -b -o ${outname%.*}.bam ${outname}
samtools sort -o ${outname%.*}_sorted.bam -T TEMP --threads 36 ${outname%.*}.bam
samtools index ${outname%.*}_sorted.bam
##############################################################################
Megablast to NCBI NT
/work/gif/remkv6/Toth/12_Bombus_dahlbomii/14_Blobtools
fasta-splitter.pl --n-parts 20 ConcatDahlbomiiGenome.FINAL.fasta
for f in *part*fasta; do echo "sh runMegablast2nt.sh "$f;done >blasts.sh
#!/bin/bash
#wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/taxdb.tar.gz
#tar -zxvf taxdb.tar.gz
#module load blast-plus
FASTA="$1"
blastn \
-task megablast \n
-query ${FASTA} \
-db /work/LAS/BioDatabase/BLASTdb/NCBI/Archives/Current/nt \
-outfmt '6 qseqid staxids bitscore std sscinames sskingdoms stitle' \
-culling_limit 10 \
-num_threads 36 \
-evalue 1e-3 \
-out ${FASTA%.**}.vs.nt.cul10.1e3.megablast.out
Megablast ouput of top best hit
HiC_scaffold_1 30195 8.396e+05 HiC_scaffold_5 OU342930.1 77.816 1464352 254517 44864 1576486 2979461 12199451 13654846 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_10 30195 8.337e+05 HiC_scaffold_5 OU342930.1 77.755 1465226 254105 45171 1487581 2890829 12199451 13654816 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_11 65598 1.236e+05 HiC_scaffold_11 HG995280.1 96.275 75837 1995 297 7573037 7648548 7502796 7578127 0.0 Bombus pascuorum Eukaryota Bombus pascuorum genome assembly, chromosome: 13
HiC_scaffold_12 65598 1.569e+05 HiC_scaffold_12 HG995277.1 97.139 93349 1981 193 8110585 8203593 7135049 7042051 0.0 Bombus pascuorum Eukaryota Bombus pascuorum genome assembly, chromosome: 10
HiC_scaffold_13 30195 8.330e+05 HiC_scaffold_5 OU342930.1 77.744 1464740 254697 45184 1492643 2895399 12199451 13654872 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_14 30195 8.323e+05 HiC_scaffold_5 OU342930.1 77.735 1464960 254678 44806 1467391 2870452 12199453 13654816 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_15 65598 1.397e+05 HiC_scaffold_15 HG995279.1 97.155 83013 1850 162 5203366 5286170 5916411 5999119 0.0 Bombus pascuorum Eukaryota Bombus pascuorum genome assembly, chromosome: 12
HiC_scaffold_16 30201 2.684e+05 HiC_scaffold_16 OU443163.1 79.008 420201 67792 12639 7096155 7499262 2048536 2465412 0.0 Bombus sylvestris Eukaryota Bombus sylvestris genome assembly, chromosome: 23
HiC_scaffold_17 30195 8.316e+05 HiC_scaffold_5 OU342930.1 77.725 1465161 254901 44829 1313971 2717408 12199453 13654875 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_18 30195 97522 HiC_scaffold_18 OU342933.1 79.851 140541 22765 2366 3697458 3833166 5531383 5671202 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 13
HiC_scaffold_19 77635 6613 HiC_scaffold_19 CP062939.1 89.308 5387 421 108 77385 82679 2491536 2496859 0.0 Bifidobacterium subtile Bacteria Bifidobacterium subtile strain KCTC 3272 chromosome, complete genome
HiC_scaffold_2 30195 8.389e+05 HiC_scaffold_5 OU342930.1 77.811 1464372 254301 44859 1556250 2958940 12199451 13654875 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_20 30194 3363 HiC_scaffold_20 OV883986.1 70.383 22838 5315 1245 153891 175970 175833 153687 0.0 Bombus pratorum Eukaryota Bombus pratorum genome assembly, chromosome: 4
HiC_scaffold_21 2575261 3297 HiC_scaffold_21 MK770119.1 76.065 6622 1422 137 38002 44539 40324 33782 0.0 Pantoea phage vB_PagS_AAS21 Viruses Pantoea phage vB_PagS_AAS21, complete genome
HiC_scaffold_22 77635 3522 HiC_scaffold_22 CP062939.1 76.201 7034 1464 180 26126 33050 2177205 2170273 0.0 Bifidobacterium subtile Bacteria Bifidobacterium subtile strain KCTC 3272 chromosome, complete genome
HiC_scaffold_23 1603886 1463 HiC_scaffold_23 CP062948.1 82.360 1695 287 11 61687 63373 382523 380833 0.0 Bifidobacterium lemurum Bacteria Bifidobacterium lemurum strain DSM 28807 chromosome, complete genome
HiC_scaffold_24 33905;1254439 8684 HiC_scaffold_24 LR698979.1 82.969 9753 1526 100 6404 16096 1767098 1776775 0.0 Bifidobacterium thermophilum;Bifidobacterium thermophilum RBL67 Bacteria Bifidobacterium thermophilum isolate MGYG-HGUT-02334 genome assembly, chromosome: 1
HiC_scaffold_25 2170413 1070 HiC_scaffold_25 BK058804.1 79.293 1555 302 19 29677 31221 27374 25830 0.0 Caudoviricetes sp. Viruses MAG TPA_asm: Siphoviridae sp. isolate ctatL78, partial genome
HiC_scaffold_26 77635 1330 HiC_scaffold_26 CP062939.1 84.996 1313 193 4 14172 15482 1575111 1576421 0.0 Bifidobacterium subtile Bacteria Bifidobacterium subtile strain KCTC 3272 chromosome, complete genome
HiC_scaffold_27 2020965 1424 HiC_scaffold_27 CP071591.1 87.066 1268 154 9 55504 56766 1997930 1996668 0.0 Bifidobacterium imperatoris Bacteria Bifidobacterium imperatoris strain JCM 32708 chromosome
HiC_scaffold_28 1682 1216 HiC_scaffold_28 CP062951.1 78.239 1976 374 49 37505 39447 2033015 2031063 0.0 Bifidobacterium longum subsp. infantis Bacteria Bifidobacterium longum subsp. infantis strain JCM 11347 chromosome, complete genome
HiC_scaffold_29 2170413 665 HiC_scaffold_29 MN855933.1 80.154 912 164 15 33439 34346 4539 5437 0.0 Caudoviricetes sp. Viruses MAG: Siphoviridae sp. isolate 66, complete genome
HiC_scaffold_3 30195 8.369e+05 HiC_scaffold_5 OU342930.1 77.829 1458484 252913 44367 1587011 2984197 12199451 13648782 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_30 42906 494 HiC_scaffold_30 MT039150.1 89.922 387 33 6 30562 30945 3442 3825 5.63e-133 Serratia entomophila Bacteria Serratia entomophila strain AGR_345 plasmid unnamed3, complete sequence
HiC_scaffold_31 30191 1731 HiC_scaffold_31 OU427027.1 73.665 5111 1074 247 1 4974 108828 113803 0.0 Bombus hypnorum Eukaryota Bombus hypnorum genome assembly, chromosome: 8
HiC_scaffold_32 65598 4348 HiC_scaffold_32 HG995279.1 82.419 5011 853 28 1 4999 3703618 3698624 0.0 Bombus pascuorum Eukaryota Bombus pascuorum genome assembly, chromosome: 12
HiC_scaffold_4 30195 8.357e+05 HiC_scaffold_5 OU342930.1 77.777 1465064 253960 44961 1515797 2918912 12199451 13654846 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_5 30195 8.396e+05 HiC_scaffold_5 OU342930.1 77.816 1464352 254517 44864 1576486 2979461 12199451 13654846 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_6 30195 8.343e+05 HiC_scaffold_5 OU342930.1 77.753 1464541 255202 44976 1506560 2909665 12199451 13654816 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 10
HiC_scaffold_7 30201 3.281e+05 HiC_scaffold_7 OU443163.1 81.014 423439 71257 4891 16360989 16777126 2070630 2492233 0.0 Bombus sylvestris Eukaryota Bombus sylvestris genome assembly, chromosome: 23
HiC_scaffold_8 30195 2.485e+05 HiC_scaffold_8 OU342938.1 77.163 436580 93923 4822 4642707 5076045 985559 1419600 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 18
HiC_scaffold_9 30195 2.083e+05 HiC_scaffold_9 OU342933.1 81.990 248578 41778 2209 2395811 2642811 5634571 5387409 0.0 Bombus terrestris Eukaryota Bombus terrestris genome assembly, chromosome: 13
Blobtools
/work/gif/remkv6/Toth/12_Bombus_dahlbomii/14_Blobtools
#9 of the pseudomolecules were taking a long time. Since we know that those are Dahlbomii, I will just substitute the blast hits to bombus from scaffold_5
cat <(head -n 9 AllBlasts.tab) AllBlasts.tab >FixedAllBlasts.tab
module load singularity;module load blobtools2;
singularity shell /opt/rit/singularity/images/blobtools2/2.2.0/blobtools2.simg
cp -rf /work/gif3/masonbrink/Serb/08_ContigElimination/01_Blobtools/taxdump/ .
ln -s ../13_ReAnnotate/ConcatDahlbomiiGenome.FINAL_Passerformes_Busco/run_hymenoptera_odb10/full_table.tsv
blobtools create --fasta ConcatDahlbomiiGenome.FINAL.fasta --cov 30_537121167.hifi_reads.fastq_ConcatDahlbomiiGenome.FINAL_minimap2_sorted.bam --busco full_table.tsv --hits FixedAllBlasts.tab --taxdump taxdump test
#connect to novaDTN through my pc's terminal
/work/gif/remkv6/Olsen/Bison/08_Blobtools/03_Blobenate
module load singularity;module load blobtools2;
singularity shell /opt/rit/singularity/images/blobtools2/2.2.0/blobtools2.simg
blobtools view --interactive test
Final plot after filtering contaminating scaffolds
Output from blobtols
"sel","_id","gc","length","30_537121167.hifi_reads.fastq_ConcatDahlbomiiGenome.FINAL_minimap2_sorted_cov","bestsumorder_phylum","id"
"","0","0.3688","32487547","27.5395","Arthropoda","HiC_scaffold_1"
"","1","0.3525","24216648","25.1137","Arthropoda","HiC_scaffold_2"
"","2","0.3563","19734131","25.1221","Arthropoda","HiC_scaffold_3"
"","3","0.3661","19374274","26.1943","Arthropoda","HiC_scaffold_4"
"","4","0.3644","18741811","24.9687","Arthropoda","HiC_scaffold_5"
"","5","0.3518","18407461","25.2911","Arthropoda","HiC_scaffold_6"
"","6","0.3762","17299723","25.66","Arthropoda","HiC_scaffold_7"
"","7","0.3667","16961761","26.6204","Arthropoda","HiC_scaffold_8"
"","8","0.3699","15367549","27.113","Arthropoda","HiC_scaffold_9"
"","9","0.3669","13970555","27.5592","Arthropoda","HiC_scaffold_10"
"","10","0.3754","13034363","27.5943","Arthropoda","HiC_scaffold_11"
"","11","0.3882","12441523","28.7737","Arthropoda","HiC_scaffold_12"
"","12","0.3673","12007047","26.8592","Arthropoda","HiC_scaffold_13"
"","13","0.3658","11734535","31.3478","Arthropoda","HiC_scaffold_14"
"","14","0.3861","11609214","26.4285","Arthropoda","HiC_scaffold_15"
"","15","0.361","7505742","24.687","Arthropoda","HiC_scaffold_16"
"","16","0.352","5244765","86.2438","Arthropoda","HiC_scaffold_17"
"","17","0.3798","3846188","23.5803","Arthropoda","HiC_scaffold_18"
"","18","0.5466","228834","5.1966","Actinobacteria","HiC_scaffold_19"
"","19","0.4487","178907","7.4422","Arthropoda","HiC_scaffold_20"
"","20","0.393","113038","21.8358","Uroviricota","HiC_scaffold_21"
"","21","0.5598","108254","4.436","Actinobacteria","HiC_scaffold_22"
"","22","0.5339","103634","4.5048","Actinobacteria","HiC_scaffold_23"
"","23","0.5584","91059","5.1736","Actinobacteria","HiC_scaffold_24"
"","24","0.5537","86719","3.8782","Uroviricota","HiC_scaffold_25"
"","25","0.5381","70448","4.5279","Actinobacteria","HiC_scaffold_26"
"","26","0.5313","59575","4.5306","Actinobacteria","HiC_scaffold_27"
"","27","0.5983","39470","4.1672","Actinobacteria","HiC_scaffold_28"
"","28","0.4656","39391","44.1131","Uroviricota","HiC_scaffold_29"
"","29","0.413","33372","1.5051","Proteobacteria","HiC_scaffold_30"
"","30","0.435","5000","0","Arthropoda","HiC_scaffold_31"
"","31","0.276","5000","8.9968","Arthropoda","HiC_scaffold_32"
Kept scaffolds 1-18,20 and 32, as the others were contaminants or lacked coverage (scaffold_31).
GC Length Coverage bestsumorder_phylum ID
0.393 113038 21.8358 Uroviricota HiC_scaffold_21
0.5537 86719 3.8782 Uroviricota HiC_scaffold_25
0.4656 39391 44.1131 Uroviricota HiC_scaffold_29
3 contigs at a length of 239,148
GC Length Coverage bestsumorder_phylum ID
0.5466 228834 5.1966 Actinobacteria HiC_scaffold_19
0.5598 108254 4.436 Actinobacteria HiC_scaffold_22
0.5339 103634 4.5048 Actinobacteria HiC_scaffold_23
0.5584 91059 5.1736 Actinobacteria HiC_scaffold_24
0.5381 70448 4.5279 Actinobacteria HiC_scaffold_26
0.5313 59575 4.5306 Actinobacteria HiC_scaffold_27
0.5983 39470 4.1672 Actinobacteria HiC_scaffold_28
0.413 33372 1.5051 Proteobacteria HiC_scaffold_30
8 contigs at a length of 734,646
Bifidobacterium accidental integration into HiC_scaffold_2
HiC_scaffold_2:24097917-24216648
awk '/^[^>]/ {total += length($0); gc += gsub(/[GC]/, "", $0)} END {printf "GC percent: %.2f%%\n", (gc / total) * 100}' BacterialInsertion.fasta
GC percent: 54.91%
So 9 contigs totaling 853,377 bp were of bacterial origin
Final Table of Contamination
| ID | GC | Length | Coverage | Best Sum Order Phylum | Best Blast Hit Genus Species |
|---|---|---|---|---|---|
| HiC_scaffold_21 | 0.393 | 113038 | 21.8358 | Uroviricota | Pantoea phage vB_PagS_AAS21 |
| HiC_scaffold_25 | 0.5537 | 86719 | 3.8782 | Uroviricota | Caudoviricetes |
| HiC_scaffold_29 | 0.4656 | 39391 | 44.1131 | Uroviricota | Caudoviricetes |
| HiC_scaffold_19 | 0.5466 | 228834 | 5.1966 | Actinobacteria | Bifidobacterium subtile |
| HiC_scaffold_22 | 0.5598 | 108254 | 4.436 | Actinobacteria | Bifidobacterium subtile |
| HiC_scaffold_23 | 0.5339 | 103634 | 4.5048 | Actinobacteria | Bifidobacterium lemurum |
| HiC_scaffold_24 | 0.5584 | 91059 | 5.1736 | Actinobacteria | Bifidobacterium thermophilum |
| HiC_scaffold_26 | 0.5381 | 70448 | 4.5279 | Actinobacteria | Bifidobacterium subtile |
| HiC_scaffold_27 | 0.5313 | 59575 | 4.5306 | Actinobacteria | Bifidobacterium imperatoris |
| HiC_scaffold_28 | 0.5983 | 39470 | 4.1672 | Actinobacteria | Bifidobacterium longum |
| HiC_scaffold_30 | 0.413 | 33372 | 1.5051 | Proteobacteria | Serratia entomophila |
| HiC_scaffold_2_split | 0.5491 | 118731 | N/A | Actinobacteria | Bifidobacterium bifidum |