HERA is a local assembly tool using assembled contigs and self-corrected long reads as input. HERA is highly efficient using SMS data to resolve repeats, which enables the assembly of highly contiguous genomes. With the help of BioNano genome maps and chromosomal anchoring information, HERA can generate ultra-long, even chromosome-scale, contigs.
It is important to note that even though HERA can be used to improve the sequence contiguity of highly heterozygous genomes, it require HiC data (and better also with BioNano data) to resolve the haplotype sequences. A new pipeline to do this is being developed but it is not included here.
Assume the software HERA is under directory HERA. All the executable files of HERA package should be put in HERA/bin.
The running of HERA requires a few other software programs.
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Downloading and installing bwa-0.7.10
git clone https://github.com/lh3/bwa.git
cd bwa; make. -
Downloading and installing DALIGNER
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Downloading and installing DAZZ_DB
Step 0: Correct the noisy long reads by CANU and finish genome assembly by CANU or MECAT, or FALCON or other assemblers to generate contigs with high sequence accuracy.
Before running HERA, you need to create a config file template. HERA provides two kinds of running patterns for connecting the whole-genome assembled contigs and filling the gaps between the paired contigs with or without the BioNano maps.
The template looks like
############################### the parameters that can be changed by users ##########################################
#the genome name(less than 5 words)
genome_name=Human
#the whole genome assembled contigs or scaffolds
genome_seq=
#the corrected long reads
Corrected_Pacbio=
#the enzyme used to form the bionano map(if no bionano map, neglecting this)
Enzyme=
#the software position
Working_Script=~/home/
#the queue used to bsub jobs
queue=
#the DAZZ_DB software position
DAZZ_DB=~/home/software/DAZZ_DB-master/
#the DALIGNER software position
DALIGNER=~/home/software/DALIGNER-master/
#the positions apart from start or end
InterIncluded_Side=25000
#internal pacbios and contigs
InterIncluded_Identity=99;
InterIncluded_Coverage=99;
#the pacbios selected for starting and ending
MinIdentity=97
MinCoverage=90
MinLength=5000
#the conditions used to filter the overlap used to construct the graph
MinIdentity_Overlap=97
MinOverlap_Overlap=1000
MaxOverhang_Overlap=100
MinExtend_Overlap=1000
#the min num path for contig pairs
MinPathNum=3
#the conditons used to merge the supercontigs and non-scaffolded contigs
MinIdentity_Merge=98
MinOverlap_Merge=10000
MaxOverhang_Merge=200
############################### end of resetting parameters ##################################################you need to fill and modify the relevant information, such as the whole genome assembled contigs or scaffold and the self-corrected long reads.
$ sh pipeline.shUsers need to note that HERA currently only supports LSF job scheduling system, and we are trying to adapt HERA to different types of cluster job systems. While users can manually modify the configuration section of job system in scripts of "04-Qsub-Mapping2Ctg.pl", "08-qsub_job_index.pl", "21-Daligner_New.pl" and "09-Qsub-Pair_Alignment.pl" to fit your job system. It should be emphasized here that after the modification, users need to ensure the uniformaity of the way HERA submits and monitors jobs .
Example like
#BSUB -J $genome-Pair-$i-$j
#BSUB -o $count.out
#BSUB -n 1
#BSUB -q $queueAfter the successful submission of pipeline.sh, HERA will take a few steps to get the reassembled genome sequences with the name of "genome_name-Final_Genome_HERA.fasta". HERA mainly includes the following five parts:
- Mapping the corrected pacbio long reads to the whole genome assembled contigs;
- Filtering the corrected pacbio long reads which are used to assemble the contigs;
- Constructing the Contig-Reads and Reads-Reads overlaping graph;
- Traversing the overlapping graph taking the contig nodes as start and end to find the connecting paths;
- Constructing and traversing the contig-to-contig path graph to define the order and orientation of the contigs;
- Constructing the consensus sequence to fill the gap and produce the final genome.
Finally, the users can get the super-contig genome and the connection information by HERA in the 06-Daligner/Selected_Path.txt and 06-Daligner/Ctg_Position.txt.
$ ll -rth ./06-Daligner/
SuperContig_Part_Info.txt
SuperContig.fasta
Selected_Path.txt
Ctg_Position.txt#Make the working dirs
mkdir 01-Pacbio_And_NonScaffold
mkdir 02-Pacbio-Alignment
mkdir 03-Pacbio-SelfAlignment
mkdir 04-Graphing
mkdir 05-PathContig
mkdir 06-Daligner
mkdir 07-FilledGap
mkdir 08-PathContig_Consensus
mkdir 09-ReAssembly
#convert the fasta to lines
$Working_Script/readstoline $genome_seq $genome_name-Genome.fasta C
#split the sequences into two files with large contigs and small contigs
$Working_Script/01-Filter_Raw_Contig_By_Length $genome_name-Genome.fasta Large_Contig.fasta Small_Contig.fasta 150000 15000
#covert the fasta formate to lines
$Working_Script/readstoline $Corrected_Pacbio $genome_name-CorrectedPacbio.fasta P
Corrected_Pacbio=$genome_name-CorrectedPacbio.fasta
#Merge the non-scaffolded contig with corrected pacbio and they are all used to construct overlaping graph
cat $Bionano_NonScaffolded_Contig $Corrected_Pacbio >Query_Merged.fasta
#Change the dir of working
cd ./01-Pacbio_And_NonScaffold
#Split the corrected pacbios and non-scaffolded contigs into parts
$Working_Script/03-fasta-splitter --n-parts 100 ../Query_Merged.fasta
#Make the list of split sequence
cd -
ls ./01-Pacbio_And_NonScaffold/*.fasta >list_Split.txt
#Make the index of Contig
bwa index $Bionano_Scaffolded_Contig
#Align the corrected pacbios and non-scaffolded contigs to scaffolded contigs
perl $Working_Script/04-Qsub-Mapping2Ctg.pl list_Split.txt $Bionano_Scaffolded_Contig ./02-Pacbio-Alignment $Working_Script $queue $genome_name >log
#Wait until the end of all alignment
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Map" |awk '{count=count+1;}END{print count;}'`;
sleep 20;
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Map" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
sleep 20;
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Map" |awk '{count=count+1;}END{print count;}'`;
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'|grep "$genome_name-Map" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
#Remove the log and pbs files
rm -f *.o*
rm -f [0-9]*.pbs
#Merge all alignment into an single file
cat ./02-Pacbio-Alignment/Part_Alignment_*.txt > ./02-Pacbio-Alignment/Total_Alignment.txt
#rm -f ./02-Pacbio-Alignment/Part_Alignment_*.txt
#Remove the pacbios and non-scaffolded contigs aligned to the internal scaffolded contigs
$Working_Script/05-Filtered_InterIncluded_Pacbio ./02-Pacbio-Alignment/Total_Alignment.txt ./02-Pacbio-Alignment/InterIncluded_Pacbio.txt $InterIncluded_Identity $InterIncluded_Coverage $InterIncluded_Side
#Record the pacbio alignment of contig's head and end
$Working_Script/06-Extract_Contig_Head_Tail_Pacbio_Alignment -Align=./02-Pacbio-Alignment/Total_Alignment.txt -MinIden=$MinIdentity -MinCov=$MinCoverage -HTLen=$InterIncluded_Side -MinLen=$MinLength
#Change the aligned positions into positive chain
$Working_Script/10-Switch_Locus_To_Positive Contig_Head_Tail_Pacbio.txt ./04-Graphing/Contig_Head_Tail_Pacbio_Pos.txt
#Extract the sequence of corrected pacbio and non-scaffoled contigs which are nonaligned or aligned to the start or end of the contigs
$Working_Script/07-extract_fasta_seq_by_name ./02-Pacbio-Alignment/InterIncluded_Pacbio.txt ./Query_Merged.fasta ./02-Pacbio-Alignment/Both_Side_Pacbio.fasta
#Split the remained pacbio or contigs into parts
cd ./03-Pacbio-SelfAlignment
$Working_Script/03-fasta-splitter --n-parts 30 ../02-Pacbio-Alignment/Both_Side_Pacbio.fasta
#Make index for every part of the pacbios and non-scaffolded contigs
cd -
ls ./03-Pacbio-SelfAlignment/*.fasta >list_outer_pacbio.txt
perl $Working_Script/08-qsub_job_index.pl list_outer_pacbio.txt $queue $genome_name>>log
#Wait until the end of making all index
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-INDEX" |awk '{count=count+1;}END{print count;}'`
sleep 20
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-INDEX" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
sleep 20;
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-INDEX" |awk '{count=count+1;}END{print count;}'`
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-INDEX" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
rm -f *.o*
rm -f [0-9]*.pbs
sleep 10;
#Align the corrected pacbios and non-scaffolded contigs to each other for finding overlaps
perl $Working_Script/09-Qsub-Pair_Alignment.pl list_outer_pacbio.txt $Working_Script $queue $genome_name >>log
#Wait until the end of making all index
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Pair" |awk '{count=count+1;}END{print count;}'`
sleep 20
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Pair" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
sleep 30
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Pair" |awk '{count=count+1;}END{print count;}'`
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-Pair" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
rm -f *.o*
rm -f [0-9]*.pbs
#Merge all alignment into an single file
cat ./03-Pacbio-SelfAlignment/Part_SelfAlignment_*.txt > ./03-Pacbio-SelfAlignment/Total_SelfAlignment.txt
#Filter the alignment for overlaping
$Working_Script/11-PacbioAlignmentFilter ./03-Pacbio-SelfAlignment/Total_SelfAlignment.txt $MaxOverhang_Overlap $MinIdentity_Overlap $MinOverlap_Overlap $MinExtend_Overlap > ./04-Graphing/PacbioAlignmentFiltered.txt
#Find the proper overlap for constructing the graph
$Working_Script/12-PacbioAlignmentLinker ./04-Graphing/PacbioAlignmentFiltered.txt $MaxOverhang_Overlap $MinExtend_Overlap > ./04-Graphing/PacbioAlignmentLinked.txt
#Constrct graph by the alignment of pacbios, and the nodes are pacbios and the edges are overlaps.
#Then Finding Contigs Pathway with the Correct Orientatios
cd ./04-Graphing/
$Working_Script/Selected_Best_Pairs PacbioAlignmentLinked.txt PacbioAlignmentLinked_BestMatch.txt
$Working_Script/13-Graph_By_Finding_Best_MaxExtending_Random_Path PacbioAlignmentLinked_BestMatch.txt >check
#Output the uniq path
cat ctg_clusters.txt |sort |uniq > ../05-PathContig/ctg_clusters_uniq.txt
cat cluster_ori.txt |sort |uniq > ../05-PathContig/cluster_ori_uniq.txt
cd -
cd 05-PathContig
#Make the corrected pacbios and non-scaffolded contigs into a line
$Working_Script/14-make_ctg_line cluster_ori_uniq.txt cluster_ori_same_chain.txt
$Working_Script/18-compute_fasta_file_len ../Query_Merged.fasta Query_Len.txt
#Change the path into the same chain of bionano scaffolds
$Working_Script/15-make_junction_by_pos ../04-Graphing/ctg_pairs.txt Query_Len.txt cluster_ori_same_chain.txt cluster_ori_same_chain_pos.txt
#Extract the aligned information of pacbios for final pathcontigs
$Working_Script/16-extract_ctg_infor_for_seq cluster_ori_same_chain_pos.txt cluster_ori_same_chain_pos_for_seq.txt
echo ">NA" >NA.fasta
echo "ATCG" >>NA.fasta
#Output the final contigs of path used to fill the gap of bionano
$Working_Script/17-extract_seq_by_pos cluster_ori_same_chain_pos_for_seq.txt ../Query_Merged.fasta NA.fasta PathContig.fasta
#Compute the length of pathcontigs
$Working_Script/18-compute_fasta_file_len PathContig.fasta ../06-Daligner/PathContig_Len.txt
##########
cd -
#make the working dirs
mkdir 10-Contig_Pairs
cd 10-Contig_Pairs
touch overlap.txt
#formating the contig pairs based on the paths
$Working_Script/03-Formate_Contig_Pairs_By_Paths overlap.txt ../05-PathContig/ctg_clusters_uniq.txt Contig_Pairs.txt
cat Contig_Pairs.txt |awk '{if($5>='$MinPathNum' && $6>='$MinPathNum' && $7>='$MinPathNum'){$8=$5+$6/3+$7/6;print $0;}}' >Contig_Pairs_Filtered.txt
#selecting the final contig pairs with clustering based on scores
$Working_Script/05-Merge_With_HighestScore_To_Sequence_By_Path Contig_Pairs_Filtered.txt ../Large_Contig.fasta SuperContig.fasta >Selected_Pairs.txt
cd -
cd 06-Daligner
#extract the paths which connects the final selected contigs
$Working_Script/19-Path2Scaffold_NoBioNano ../10-Contig_Pairs/Selected_Pairs.txt ../05-PathContig/ctg_clusters_uniq.txt PathContig_Len.txt Path_Scaffold.txt
#rename the path contigs
$Working_Script/20-PathContig-Rename_NoBioNano Path_Scaffold.txt ../05-PathContig/PathContig.fasta PathContig_Rename.fasta >log
$Working_Script/Rename1 ../10-Contig_Pairs/SuperContig.fasta SuperContig_Rename.fasta >Rename_Pairs.txt
$Working_Script/Rename2 Rename_Pairs.txt PathContig_Rename.fasta PathContig_Rename2.fasta
mv -f PathContig_Rename2.fasta PathContig_Rename.fasta
#formating the connected scaffold
$Working_Script/01-Gap_Count SuperContig_Rename.fasta $Enzyme Gap.txt
$Working_Script/01-Finding_Contigs_Gap Gap.txt Scaffold2Ctg_Gap.txt
$Working_Script/02-Split_Scaffold_To_Contigs SuperContig_Rename.fasta Prosudo_ScaffoldNonEnzyme2Contig.fasta $Enzyme
#aligning the path-contigs to scaffold
perl $Working_Script/21-Daligner_New.pl Scaffold2Ctg_Gap.txt Prosudo_ScaffoldNonEnzyme2Contig.fasta PathContig_Rename.fasta $queue qsub $Working_Script $genome_name $DAZZ_DB $DALIGNER
#Wait until the end of all alignment
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-DALIGNER" |awk '{count=count+1;}END{print count;}'`;
sleep 20;
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-DALIGNER" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
sleep 20;
job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-DALIGNER" |awk '{count=count+1;}END{print count;}'`;
while (($job>=1))
do job=`bjobs -w|awk '{if($4=="'$queue'")print $0;}'| grep "$genome_name-DALIGNER" |awk '{count=count+1;}END{print count;}'`;echo $job;sleep 20;
done
#Remove the log and pbs files
rm -f *.o*
rm -f [0-9]*.pbs
#filling the gaps with the path-contigs
$Working_Script/22-Filling-Gap Scaffold2Ctg_Gap.txt Prosudo_ScaffoldNonEnzyme2Contig.fasta PathContig_Rename.fasta SuperContig.fasta
#formating the final genome
cat SuperContig.fasta ../$Bionano_NonScaffolded_Contig |awk 'BEGIN{count=1;}{if($0~/^>/){print ">SuperContig"count"END";count++;}else{print $0;}}' >../$genome_name-Final_Genome_HERA.fastaHERA is highly efficient for generating highly contiguous and complete or nearly complete sequences for small genomes such as fungi as well as homozygous genomes. HERA may be applied to a genome for several rounds to get desired results. For highly heterozygous genomes, a lot of manual work may be required.
Du, H., Liang, C. (2018). Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads. bioRxiv doi: https://doi.org/10.1101/345983
The detailed usage is described in the man page available together with the source code. If you have questions about HERA, you may send the questions to [email protected] or [email protected].