Chris:LabNotes/Moleculo/2013/2013-5-15

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Moleculo Data Analysis Part 2[edit]

1a-c_Illumina_Test_Assembly[edit]

  • Triton folder: </projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/>
  • We are going to now run SOAPdenovo2 assembly on corrected read files
  • In order to do this, we are going to test three different conditions for assembly (each found in their consecutive folders):
    • <1a_Illumina_Test_Assembly_Control>: Assembly using only non-corrected original reads
    • <1b_Illumina_Test_Assembly_no-Mate-correction>: Assembly using corrected 200bp reads with no correction on mate-paired 2kb reads (this is the protocol used in Assemblathon2 in which the reads used to scaffold are not corrected)
    • <1c_Illumina_Test_Assembly_all-correction>: Assembly using only corrected reads for both 200bp and 2kb mate-paired read files
  • The steps for correction: (specifically for 1a_Illumina_Test_Assembly_Control)

1) Make config files for pregraph, map, and GapCorrect

  • Pregraph config file <test_assembly_control.cfg>
    • Used parameters similar to Assemblathon2
    • Key feature is that for both libraries, we are specifying the original read files (q1,q2) instead of the paired corrected file
#maximal read length
max_rd_len=200
[LIB]
name=HB_gDNA200b_121224L1-4_filtered
#average insert size
avg_ins=200
#if sequence needs to be reversed 
reverse_seq=0
#in which part(s) the reads are used
asm_flags=3
#in which order the reads are used while scaffolding
rank=1
# cutoff of pair number for a reliable connection (default 3)
pair_num_cutoff=3
q1=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R1_filtered.fastq.gz
q2=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R2_filtered.fastq.gz
[LIB]
name=HB_gDNA2kb_NoIndex_L005_001_filtered
avg_ins=2000
reverse_seq=1
asm_flags=2
rank=2
pair_num_cutoff=5
map_len=35
q1=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R1_001_filtered.fastq.gz
q2=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R2_001_filtered.fastq.gz
  • Map config file <test_assembly_control.map.cfg>
    • Same as the pregraph config file, just without the max read length parameter
[LIB]
name=HB_gDNA200b_121224L1-4_filtered
#average insert size
avg_ins=200
#if sequence needs to be reversed 
reverse_seq=0
#in which part(s) the reads are used
asm_flags=3
#in which order the reads are used while scaffolding
rank=1
# cutoff of pair number for a reliable connection (default 3)
pair_num_cutoff=3
q1=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R1_filtered.fastq.gz
q2=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R2_filtered.fastq.gz
[LIB]
name=HB_gDNA2kb_NoIndex_L005_001_filtered
avg_ins=2000
reverse_seq=1
asm_flags=2
rank=2
pair_num_cutoff=5
map_len=35
q1=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R1_001_filtered.fastq.gz
q2=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA2kb_NoIndex_L005_R2_001_filtered.fastq.gz
  • Gap Closer config file <test_assembly_control.GC.cfg>
    • Only need to include library for 200bp reads (no need for scaffolding reads)
[LIB]
name=HB_gDNA200b_121224L1-4_filtered
#average insert size
avg_ins=200
#if sequence needs to be reversed
reverse_seq=0
#in which part(s) the reads are used
asm_flags=3
#in which order the reads are used while scaffolding
rank=1
# cutoff of pair number for a reliable connection (default 3)
pair_num_cutoff=3
q1=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R1_filtered.fastq.gz
q2=/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Illumina_raw/HB_gDNA200b_121224L1-4_R2_filtered.fastq.gz

2) Write commands for assembly (ass.sh)

  • Here, we are writing commands for assembly in separate command lines (in order to better control each part of assembly)
    • Need commands for pregraph, contig, map, and scaff
/home/cjwei/software/SOAPdenovo2-src-r223/standardPregraph/SOAPdenovo-63mer pregraph -s /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control.cfg -d 1 -p 32 -K 29 -o /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/pregraph.log
/home/cjwei/software/SOAPdenovo2-src-r223/standardPregraph/SOAPdenovo-63mer contig -g /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control -M 3 >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/contig.log
/home/cjwei/software/SOAPdenovo2-src-r223/standardPregraph/SOAPdenovo-63mer map -s /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control.map.cfg -g /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control -p 32 >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/map.log
/home/cjwei/software/SOAPdenovo2-src-r223/standardPregraph/SOAPdenovo-63mer scaff -g /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control -b 1.2 -F -p 32 > /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/scaff.log

3) Write command for GapCloser <GapCloser.sh>

  • This is performed after assembly and scaffolding
/home/cjwei/software/SOAPdenovo2-src-r223/tools/GapCloser -a /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control.scafSeq -b /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control.GC.cfg -l 200 -o /projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/test_assembly_control.scafSeq.GC -p 29 -t 32 >/projects/zhang-lab/cjwei/130419_Oncomelania_Ilumina_Moleculo/Hybrid_Assembly_5_14_2013/1a_Illumina_Test_Assembly_Control/GapCloser.log

4) Create 1a_control_assembly in order to submit for Triton to run

  • The steps for assembly for using corrected 200bp and/or corrected 2kb reads were modified to switch q1,q2 to p in the config files in order to switch to corrected files