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Pipeline for the linux environment
Raw reads of wheat
F:\fastqc>java -jar Trimmomatic-0.38\trimmomatic-0.38.jar SE -phred33 Leafcontro
l.fastq Leafcontroltrimmed1.fq ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILIN
G:3 SLIDINGWINDOW:4:20 MINLEN:36 HEADCROP:10 CROP:300
Check quality of reads (FastQC)
Trimming reads (remove adapter,etc.)
Filtered to Clean Reads (SolexaQA)
Mapped with genome using TopHat
Bowtie2 index file to be generated
# Merge all E. coli reference genomes into one genomes.fna file
cat ref_genomes/ecoli/*.fna > genomes.fna
# create bowtie2 index database (database name: ecoli)
bowtie2-build genomes.fna ecoli
# use 'inspect' to check the content of your database
bowtie2-inspect -n ecoli
# define your BOWTIE2_INDEXES directory and move the database files into it
export BOWTIE2_INDEXES=/path/to/my/bowtie2/databases/
cp *.bt2 $BOWTIE2_INDEXES
# Bowtie2 mapping
# map reads (sample.fastq) against the E. coli genome database 'ecoli'. database is defined by option -x
bowtie2 -x ecoli -1 SAMPLE_r1.fastq -2 SAMPLE_r2.fastq -U SAMPLE_single_reads.fastq --no-unal -p 12 -S SAMPLE.sam
/opt/software/bin/python2.7 /backup/uba/animesh/hisat2-2.1.0/extract_splice_sites.py Triticum_aestivum.IWGSC1.0+popseq.27.gtf >Triticum_aestivum.IWGSC.ss
/opt/software/bin/python2.7 /backup/uba/animesh/hisat2-2.1.0/extract_exons.py Triticum_aestivum.IWGSC1.0+popseq.27.gtf >Triticum_aestivum.IWGSC.exon
1. hisat2 index
gunzip Triticum_aestivum.IWGSC.dna.toplevel.fa.gz
Build the reference index
hisat2-build -p 20 -f Triticum_aestivum.IWGSC.dna.toplevel.fa Triticum_aestivum_ref
[uba@webapp animesh]$ {hisat2-build Triticum_aestivum.IWGSC.dna.toplevel.fa Triticum_aestivum.IW_ref
Settings:
Output files: "Triticum_aestivum.IW_ref.*.ht2l"
Total time for call to driver() for forward index: 03:43:07
2. Map the reads for each sample to the reference genome:
for i in $(ls *.fastq | rev | cut -c 10- |rev | uniq); do echo /backup/uba/animesh/hisat2-2.1.0/hisat2 -p 20 -q --dta -x /backup/uba/animesh/2_hisat2_index/Triticum_aestivum_ref -1 ${i}_R1.fastq -2 ${i}_R2.fastq -S /backup/uba/animesh/${i}.bam;done
/backup/uba/animesh/hisat2-2.1.0/hisat2 -p 20 -q --dta -x /backup/uba/animesh/2_hisat2_index/Triticum_aestivum_ref -1 /backup/uba/animesh/1_trimmed_reads/paired_PSTNWR-1_R1.fastq -2 /backup/uba/animesh/1_trimmed_reads/paired_PSTNWR-1_R2.fastq -S paired_PSTNWR-1.sam
paired_PSTNWR-1
34765376 reads; of these:
34765376 (100.00%) were paired; of these:
9482376 (27.28%) aligned concordantly 0 times
20842587 (59.95%) aligned concordantly exactly 1 time
4440413 (12.77%) aligned concordantly >1 times
----
9482376 pairs aligned concordantly 0 times; of these:
319297 (3.37%) aligned discordantly 1 time
----
9163079 pairs aligned 0 times concordantly or discordantly; of these:
18326158 mates make up the pairs; of these:
16609314 (90.63%) aligned 0 times
1041402 (5.68%) aligned exactly 1 time
675442 (3.69%) aligned >1 times
76.11% overall alignment rate
paired_PSTNWR-3
40689150 reads; of these:
40689150 (100.00%) were paired; of these:
10814608 (26.58%) aligned concordantly 0 times
23654972 (58.14%) aligned concordantly exactly 1 time
6219570 (15.29%) aligned concordantly >1 times
----
10814608 pairs aligned concordantly 0 times; of these:
2910966 (26.92%) aligned discordantly 1 time
----
7903642 pairs aligned 0 times concordantly or discordantly; of these:
15807284 mates make up the pairs; of these:
9480132 (59.97%) aligned 0 times
3197270 (20.23%) aligned exactly 1 time
3129882 (19.80%) aligned >1 times
88.35% overall alignment rate
paired_PSTDSWR-2
34345362 reads; of these:
34345362 (100.00%) were paired; of these:
6818591 (19.85%) aligned concordantly 0 times
22623254 (65.87%) aligned concordantly exactly 1 time
4903517 (14.28%) aligned concordantly >1 times
----
6818591 pairs aligned concordantly 0 times; of these:
404124 (5.93%) aligned discordantly 1 time
----
6414467 pairs aligned 0 times concordantly or discordantly; of these:
12828934 mates make up the pairs; of these:
10978230 (85.57%) aligned 0 times
1142167 (8.90%) aligned exactly 1 time
708537 (5.52%) aligned >1 times
84.02% overall alignment rate
paired_PSTDSWR-4
32999587 reads; of these:
32999587 (100.00%) were paired; of these:
10207398 (30.93%) aligned concordantly 0 times
17923952 (54.32%) aligned concordantly exactly 1 time
4868237 (14.75%) aligned concordantly >1 times
----
10207398 pairs aligned concordantly 0 times; of these:
1987096 (19.47%) aligned discordantly 1 time
----
8220302 pairs aligned 0 times concordantly or discordantly; of these:
16440604 mates make up the pairs; of these:
11770203 (71.59%) aligned 0 times
2287983 (13.92%) aligned exactly 1 time
2382418 (14.49%) aligned >1 times
82.17% overall alignment rate
3. #Sort and convert the SAM files to BAM:
samtools sort -@ 20 -o paired_PSTNWR-1.bam paired_PSTNWR-1.sam
samtools view -@ 20 -u paired_PSTNWR-1.sam | samtools sort -@ 20 -o paired_PSTNWR-1.bam
#htseq-count
4. Assemble transcripts for each sample:
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -p 30 -G /backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.41.gtf -o paired_PSTNWR-1.gtf -l paired_PSTNWR-1 paired_PSTNWR-1.bam
Assemble and estimate transcript abundances (if run before merge, will estimate abundence for each situation)
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -e -p 30 -G /backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.41.gtf -o paired_PSTNWR-1.gtf paired_PSTNWR-1.bam -A paired_PSTNWR-1.adundance -C paired_PSTNWR-1.fullycovered
generate .abundances, .fullycovered, .gtf
5. Merge transcripts from all samples:
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie --merge -p 30 -G /backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.41.gtf -o merged_paired.gtf mergelist.txt
@@@@@@@@@@@@@
cat merged_paired.gtf | grep -v "^#" | awk '$3=="transcript" {print}' | wc -l
187188
grep -c "^>" sc.fasta
Examine how the transcripts compare to the reference annotation (optional):
$ gffcompare –r chrX_data/genes/chrX.gtf –G –o mergedstringtie_merged.gtf
/backup/uba/animesh/cufflinks-2.2.1.Linux_x86_64/cuffcompare -r /backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.41.gtf -G -o merged merged_paired.gtf
output: merged.stats
merged.combined.gtf
merged.merged_paired.gtf.ref
mapmerged.loci
merged.merged_paired.gtf.tmap
merged.tracking
cat merged.stats
#= Summary for dataset: merged_paired.gtf :
# Query mRNAs : 187188 in 138813 loci (133731 multi-exon transcripts)
# (28765 multi-transcript loci, ~1.3 transcripts per locus)
# Reference mRNAs : 146518 in 120586 loci (106275 multi-exon)
# Super-loci w/ reference transcripts: 119003
#--------------------| Sn | Sp | fSn | fSp
Base level: 100.0 84.7 - -
Exon level: 101.9 88.3 100.0 90.0
Intron level: 100.0 92.6 100.0 94.5
Intron chain level: 100.0 79.5 100.0 88.9
Transcript level: 100.0 78.3 98.1 76.8
Locus level: 100.0 86.4 100.0 86.4
Matching intron chains: 106312
Matching loci: 120586
Missed exons: 0/548858 ( 0.0%)
Novel exons: 48934/633099 ( 7.7%)
Missed introns: 85/420232 ( 0.0%)
Novel introns: 25789/453965 ( 5.7%)
Missed loci: 0/120586 ( 0.0%)
Novel loci: 18702/138813 ( 13.5%)
Total union super-loci across all input datasets: 138813
@@@@@@@@@@@@@@@
6. Estimate transcript abundances and create table counts for Ballgown:
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -e -B -p 30 -G merged.combined.gtf -o ballgown_paired_PSTNWR-1.gtf paired_PSTNWR-1.bam
ballgown_paired_PSTNWR-1.gtf
ballgown_paired_PSTDSWR-2.gtf
ballgown_paired_PSTNWR-3.gtf
ballgown_paired_PSTDSWR-4.gtf
4 folder with .ctab
tctd
scsd
tcsc
tdsd
merge /backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie --merge -p 30 -G /backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.41.gtf -o tolerant_merged_paired.gtf tolerant_mergelist.txt
abundance, /backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -e -B -p 30 -G tolerant_merged_paired.gtf -o tolerant_ballgown_paired_PSTNWR-1.gtf paired_PSTNWR-1.bam
tablecount
7. Run python prepDE.py -i /backup/uba1/animesh/4_differential_expression/tctd/tctd_sample_lst.txt (server 9)
gene_count_matrix.csv
transcript_count_matrix.csv
/backup/uba/animesh/cufflinks-2.2.1.Linux_x86_64/gffread -w tc.fasta -g "/backup/uba/animesh/2_hisat2_index/Triticum_aestivum.IWGSC.dna.toplevel.fa" "/backup/uba/animesh/3_hisat2_dta/paired_PSTNWR-1.gtf"
# print from gtf file
awk -F "\t" '$3=="transcript"{print $9, $5-$4}' file.gtf > output.txt
awk -F "\t" '$3=="transcript" {print $9 "\t" $1 "\t" $4 "\t" $5 "\t" $5-$4+1}' tdsd_merged_paired.gtf >tdsd_merged_paired.txt
"/backup/uba/animesh/2. hisat2_index/Triticum_aestivum.IW_ref.2.ht2l"
/backup/uba/animesh/hisat2-2.1.0/hisat2 -x /backup/uba/animesh/Triticum_aestivum.IW_ref -1 /backup/uba/animesh/trimmed_reads/paired_PSTNWR-1_R1.fastq -2 /backup/uba/animesh/trimmed_reads/paired_PSTNWR-1_R2.fastq -S /backup/uba/animesh/hisat2_result/paired_PSTNWR-1.sam
34765376 (100.00%) were paired; of these:
9215483 (26.51%) aligned concordantly 0 times
21028587 (60.49%) aligned concordantly exactly 1 time
4521306 (13.01%) aligned concordantly >1 times
----
9215483 pairs aligned concordantly 0 times; of these:
311491 (3.38%) aligned discordantly 1 time
----
8903992 pairs aligned 0 times concordantly or discordantly; of these:
17807984 mates make up the pairs; of these:
16268120 (91.35%) aligned 0 times
938180 (5.27%) aligned exactly 1 time
601684 (3.38%) aligned >1 times
76.60% overall alignment rate
/backup/uba/animesh/hisat2-2.1.0/hisat2 -x /backup/uba/animesh/Triticum_aestivum.IW_ref -1 /backup/uba/animesh/trimmed_reads/paired_PSTDSWR-2_R1.fastq -2 /backup/uba/animesh/trimmed_reads/paired_PSTDSWR-2_R2.fastq -S /backup/uba/animesh/hisat2_result/paired_PSTDSWR-2.sam
34345362 reads; of these:
34345362 (100.00%) were paired; of these:
6554829 (19.09%) aligned concordantly 0 times
22817416 (66.44%) aligned concordantly exactly 1 time
4973117 (14.48%) aligned concordantly >1 times
----
6554829 pairs aligned concordantly 0 times; of these:
397387 (6.06%) aligned discordantly 1 time
----
6157442 pairs aligned 0 times concordantly or discordantly; of these:
12314884 mates make up the pairs; of these:
10625212 (86.28%) aligned 0 times
1047220 (8.50%) aligned exactly 1 time
642452 (5.22%) aligned >1 times
84.53% overall alignment rate
You have new mail in /var/spool/mail/uba
/backup/uba/animesh/hisat2-2.1.0/hisat2 -p 8 -x /backup/uba/animesh/Triticum_aestivum.IW_ref -1 /backup/uba/animesh/trimmed_reads/paired_PSTNWR-3_R1.fastq -2 /backup/uba/animesh/trimmed_reads/paired_PSTNWR-3_R2.fastq -S /backup/uba/animesh/hisat2_result/paired_PSTNWR-3.sam
40689150 reads; of these:
40689150 (100.00%) were paired; of these:
10501652 (25.81%) aligned concordantly 0 times
23874615 (58.68%) aligned concordantly exactly 1 time
6312883 (15.51%) aligned concordantly >1 times
----
10501652 pairs aligned concordantly 0 times; of these:
2910460 (27.71%) aligned discordantly 1 time
----
7591192 pairs aligned 0 times concordantly or discordantly; of these:
15182384 mates make up the pairs; of these:
8994504 (59.24%) aligned 0 times
3085483 (20.32%) aligned exactly 1 time
3102397 (20.43%) aligned >1 times
88.95% overall alignment rate
/backup/uba/animesh/hisat2-2.1.0/hisat2 -p 8 -x /backup/uba/animesh/Triticum_aestivum.IW_ref -1 /backup/uba/animesh/trimmed_reads/paired_PSTDSWR-4_R1.fastq -2 /backup/uba/animesh/trimmed_reads/paired_PSTDSWR-4_R2.fastq -S /backup/uba/animesh/hisat2_result/paired_PSTDSWR-4.sam
32999587 reads; of these:
32999587 (100.00%) were paired; of these:
9942903 (30.13%) aligned concordantly 0 times
18107088 (54.87%) aligned concordantly exactly 1 time
4949596 (15.00%) aligned concordantly >1 times
----
9942903 pairs aligned concordantly 0 times; of these:
1988726 (20.00%) aligned discordantly 1 time
----
7954177 pairs aligned 0 times concordantly or discordantly; of these:
15908354 mates make up the pairs; of these:
11373413 (71.49%) aligned 0 times
2185980 (13.74%) aligned exactly 1 time
2348961 (14.77%) aligned >1 times
82.77% overall alignment rate
2. Sort and convert the SAM files to BAM: Alignment data (.bam)
samtools view -bS paired_PSTNWR-1.sam -@ 30 > paired_PSTNWR-1.bam
samtools view -bS paired_PSTDSWR-2.sam -@ 30 > paired_PSTDSWR-2.bam
samtools sort -@ 15 -o "/backup/uba/animesh/4. assembly/paired_PSTNWR-1.sam" "/backup/uba/animesh/3. hisat2_result/paired_PSTNWR-1.sam"
samtools sort -@ 15 -o "/backup/uba/animesh/4. assembly/paired_PSTDSWR-2.sam" "/backup/uba/animesh/3. hisat2_result/paired_PSTDSWR-2.sam"
samtools sort -@ 20 -o "/backup/uba/animesh/4. assembly/paired_PSTNWR-3.sam" "/backup/uba/animesh/3. hisat2_result/paired_PSTNWR-3.sam" | samtools view -bS "/backup/uba/animesh/4. assembly/paired_PSTNWR-3.sam" -@ 20 > "/backup/uba/animesh/4. assembly/paired_PSTNWR-3.bam"
samtools sort -@ 8 -o ERR188044_chrX.bam ERR188044_chrX.sam
samtools view -u "/backup/uba/animesh/3. hisat2_result/paired_PSTNWR-3.sam" | samtools sort -@ 7 - -o "/backup/uba/animesh/4. assembly/paired_PSTNWR-3.bam"
samtools view -@ 20 -u "/backup/uba/animesh/3. hisat2_result/paired_PSTDSWR-4.sam" | samtools sort -@ 15 - -o "/backup/uba/animesh/4. assembly/paired_PSTDSWR-4.bam"
samtools view -bS eg2.sam > eg2.bam | samtools sort eg2.bam -o eg2.sorted.bam
3. Assemble transcripts for each sample:
#Mapping
stringtie -p 8 -G //.gtf -o //.gtf -1 //.bam
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -p 30 -G "/backup/uba/animesh/4. assembly/Triticum_aestivum.IWGSC.41.gtf" -o "/backup/uba/animesh/4. assembly/paired_PSTDSWR-4.gtf" -l "/backup/uba/animesh/3. hisat2_result/paired_PSTDSWR-4" "/backup/uba/animesh/4. assembly/paired_PSTDSWR-4.bam"
./cufflinks -p 8 --library-type fr-unstranded -o cufflinks_out_gtf -G /home/refgene.gtf /home/tophat-/accepted_hits.bam
#Merge transcripts from all samples:
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie --merge -p 20 -G "/backup/uba/animesh/4. assembly/Triticum_aestivum.IWGSC.41.gtf" -o stringtie_merged_paired.gtf mergelist.txt
#Estimate transcript abundances and create table counts for Ballgown:
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -e -B -p 8 -G stringtie_merged_paired.gtf -paired_PSTNWR-1.gtf paired_PSTNWR-1.bam
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie –e –B -p 8 -G stringtie_merged_paired.gtf -paired_PSTDSWR-2.gtf paired_PSTDSWR-2.bam
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie –e –B -p 8 -G stringtie_merged_paired.gtf -paired_PSTNWR-3.gtf paired_PSTNWR-3.bam
/backup/uba/animesh/stringtie-1.3.5.Linux_x86_64/stringtie –e –B -p 8 -G stringtie_merged_paired.gtf -paired_PSTDSWR-4.gtf paired_PSTDSWR-4.bam
#extract fasta
/backup/uba/animesh/cufflinks-2.2.1.Linux_x86_64/gffread -w transcripts.fa -g "/backup/uba/animesh/2. hisat2_index/Triticum_aestivum.IWGSC.dna.toplevel.fa" "/backup/uba/animesh/4. assembly/stringtie_merged_paired.gtf"
# how many transcripts?
185456
# transcript >200
172644
5. ORF predictor
3a. predict ORF
output is protein seq with orf
no ORF - 0 seq
with ORF (>output) - 172548 seq
172548 seq (orf sequence)
3b. orf less than 100 aa kept -- sequence greater than 300nt/100aa discarded,
17159 seq (>output+100.fasta)
3c. extractCDS from RNA seq transcript as per output+100.fasta
ORFoutput is RNAseq transcript sequence with ORF portion less than 100 aa (nt)
17159 seq (>ORFoutput)
4. coding potential calculator -- >=0.5 CPC score discarded
17158 seq with CPC < 0.5 (>CPCoutput.fasta)
5. House Keeping Genes
SILVA (SSU Ref NR 99, LSU Ref) rRNA data -- two project for SSU and LSU(195 SSU, 1712 LSU)
http://noncode.org NONCODE (current version v5.0) is an integrated knowledge database dedicated to non-coding RNAs (excluding tRNAs and rRNAs) (12979)
http://gtrnadb2009.ucsc.edu/ Genomic tRNA database (2169)
http://combio.pl/rrna/browse/ viridiplante 5SrDNA database (nucleus, mitochondria, plastid) (0)
http://greenc.sciencedesigners.com/wiki/Species:Triticum_aestivum Triticum aestivum lncRNAs (27114)
http://greengenes.secondgenome.com/ The Greengenes Database 16s rRNA GENES database and tools
6389 seq removed
makeblastdb –in alltrnas.fasta –dbtype nucl –parse_seqids
blastn –db F:\LAB\RNASeq_linux\HousekeepingGenes\new –query CPCoutput1.fasta –out lnew_trna.fasta -evalue 10 -num_threads 4 -outfmt "6 qseqid sseqid pident qlen length mismatch gapope evalue bitscore"
# compare the assembled transcripts to known transcripts
gffcompare -r Triticum_aestivum.IWGSC.41.gtf -G -o merged stringtie_merged_paired.gtf
/backup/uba1/animesh/gffcompare-0.10.8.Linux_x86_64/gffcompare -r /backup/uba1/animesh/millet/reference/icrisat/pearl_millet_v1.1.gtf -i mergelist.txt
####
/backup/uba1/animesh/gffcompare-0.10.8.Linux_x86_64/gffcompare -r /backup/uba1/animesh/millet/reference/icrisat/pearl_millet_v1.1.gtf -i mergelist.txt
/backup/uba1/animesh/stringtie-1.3.5.Linux_x86_64/stringtie -e -B -p 30 -G gffcmp.combined.gtf -o rt_ballgown.gtf /backup/uba1/animesh/millet/mapping/rcrt/Roottreatedtrimmed.bam
python prepDE.py -i sample_lst.txt
"/usr/local/bin/python2.7" prepDE.py -i sample_list.txt
perl misa.pl
samtools stats Leafcontroltrimmed.bam | grep "is sorted:"
1. Index the genome assembly (again!)
samtools faidx "/backup/uba1/animesh/millet/reference/icrisat/pearl_millet_v1.1.merged.fa"
2. Run 'mpileup' to generate VCF format
samtools mpileup -g -d8 -Q20 -f "/backup/uba1/animesh/millet/reference/icrisat/pearl_millet_v1.1.merged.fa" Leafcontroltrimmed.bam Leaftreatedtrimmed.bam Rootcontroltrimmed.bam Roottreatedtrimmed.bam > pmrawtrimmed.bcf
3. Call SNPs...
bcftools view -bvcg pmrawtrimmed.bcf > pmvartrimmed.bcf
4. Filter
RNAseq aligment measured (RScQC)
Mapped to wheat lncRNA
##Paper
Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown
https://www.nature.com/articles/nprot.2016.095
Nature Protocols volume 11, pages 1650–1667 (2016)
UClncR: Ultrafast and comprehensive long non-coding RNA detection from RNA-seq
https://www.nature.com/articles/s41598-017-14595-3
Scientific Reportsvolume 7, Article number: 14196 (2017)
Genome-wide analysis of differentially expressed lncRNAs and mRNAs in primary gonadotrophin adenomas by RNA-seq
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354857/
Oncotarget/2016
Characterization of lncRNAs involved in cold acclimation of zebrafish ZF4 cells
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195468
plosone/2018
scp -r uba@172.16.10.11:/backup/uba/animesh/3_hisat2_dta/PSTNWR-1 /drives/f/R/Wheat (from window terminal)
scp /backup/uba/animesh/3_hisat2_dta/ballgown_paired_PSTNWR-1.gtf uba1@172.16.10.9:/backup/uba1/animesh/ballgown/PSTNWR-1