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khuang28jhu / bs3

BS-Seeker3: An Ultra-fast, Versatile Pipeline for Mapping Bisulfite-treated Reads.
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alignment file is empty and no mapping #8

Open sbaheti opened 6 years ago

sbaheti commented 6 years ago

hi I am trying to use BS-seeker 3 but non of my reads are getting aligned. Here is the command and screen output

./bs3-align -1 14083.FCHTYN2BBXX_L5_R1_IAAGAGG.fastq.gz -2 14083.FCHTYN2BBXX_L5_R2_IAAGAGG.fastq.gz -g GCA_000001405.15_GRCh38_no_alt_analysis_set.fa -d $PWD/reference_genome/ -o 14083 -f bam 

 BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 30s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,426,124 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 29s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,418,803 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 29s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,154,263 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 28s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,373,748 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 29s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,324,675 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 29s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,422,459 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... 27s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,497,267 2 Welcome to SNAP version 1.0beta.23.

Loading index from directory... Welcome to SNAP version 1.0beta.23.

Loading index from directory... 28s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,300,670 2 27s. 1905073286 bases, seed size 20 Aligning. Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 2,183,328 0 (0.00%) 0 (0.00%) 0 (0.00%) 2,183,328 (100.00%) 0.00% 2,166,000 1 (None, None) (None, None) (None, None) (None, None) (None, None) (None, None) (None, None) (None, None) (None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 53383328 Number of unique-hits reads (before post-filtering): 0.0 Number of reads mapped after post-filtering 0.0

Alignment Time: 460.614155054secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0.0 Total Number of Cs in CpG context: 0.0 Total Number of Cs in CHG context: 0.0 Total Number of Cs in CHH context: 0.0

Rate of Methylation mCG 0.000% mCHG 0.000% mCHH 0.000%

khuang28jhu commented 6 years ago

From above,

"Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s) 6,400,000 0 (0.00%) 0 (0.00%) 0 (0.00%) 6,400,000 (100.00%) 0.00% 3,497,267 2"

Snap failed to align any of the reads.

How long are the reads?

Can you share with me a small subset (maybe 100 reads) of your input file

sbaheti commented 6 years ago

my original read length is 50 bp paired end but after trimming through trim galore it varies from 15-50bp long. Here is an example of the read1

@K00316:163:HTYN2BBXX:5:1101:1458:1384 1:N:0:NAGAGG CGGGTGCGGTGGTTTATGTTTGTAATTTTAGTATTTTGGGAGG + AAFJJJJJJFJJJJJJJJJJJJJJJAFJJJJAJJFJJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:1742:1384 1:N:0:NAGAGG TGGTAATTGTGTAGTTAAATTTGTTAGTTGTTGTAGTGGG + FFJJJJJJJFJAJJJJJJJJJFFJFJJFFFFJJ7FFAJJJ @K00316:163:HTYN2BBXX:5:1101:1763:1384 1:N:0:NAGAGG TGGTGGTGGGTTGTGAGTTGATGATTAGGTTTGTGATGAGT + FFJJJJFJJJAFJAJFJAFJJJJJAAFFJ7FJJFJJ7FFJ- @K00316:163:HTYN2BBXX:5:1101:2392:1384 1:N:0:NAGAGG CGGGCGCGGTGGTTGACGTTTGTAATTTTAGTTTTTCGGGAAG + AAFFJJJJFAFFAFJJJJFJJJJJJJFJJJJFFJJJJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:2859:1384 1:N:0:NAGAGG CGGTTTCGGTTTTTTAAAGTGTTGGGATTATAGGTATGAGT + FFJFJJJJJJFJJJJJJJJJJFJJJJJJJJJJJJJJJJ<FJ @K00316:163:HTYN2BBXX:5:1101:3853:1384 1:N:0:NAGAGG TGGTTTGTGTTGTTTTTTTTTTTTTTATTTTTTTTTTTTTTT + AAFFJJJJJJJJJJJJJJJJJJJJJJ<JJJJJJJJJJ-JJJJ @K00316:163:HTYN2BBXX:5:1101:4462:1384 1:N:0:NAGAGG CGGTATAGTTTTTAGAGGGATGTTAGTGTGGTTTTCGTATGA + FFJFJFJJFFAJJJJFJFJJFJJFJJJJJJJJJJJJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:6938:1384 1:N:0:NAGAGG CGGAAGATTTAAAGTATTTTGCGTAGGAAGTAGGTTTGGGT + AFFFFJJJJJJJJJJJFJ<FJJAAJJFJJJ<JJ7JFJFJFJ @K00316:163:HTYN2BBXX:5:1101:7121:1384 1:N:0:NAGAGG CGGTGGAGGGTTTTGATTATGAGTTGTTTAGTTTTTTGGTG + AAJAJJJFJA<AJ<JJF-JJJJFFAJA-FFJFFA-FAFFJJ @K00316:163:HTYN2BBXX:5:1101:7222:1384 1:N:0:NAGAGG CGGGCGTTATGGCGGGCGTTTGTAGTTTTAGTTATTCGGGAG + FAFJJJAAJFJJJJJJFJ7FJJJJJFFJJJJJJJJJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:7791:1384 1:N:0:NAGAGG TGGTTAGGTTTTATATTTAGGTGTGGGTTATTGGGGGTGTAG + AFJFJJJJJFJJJJJJJJJJJJJJJJJJFJJJJJJJJ<FAJJ @K00316:163:HTYN2BBXX:5:1101:7994:1384 1:N:0:NAGAGG CGGGTACGGTGGTTTACATTTGTAATTTTAGTATTTCGAGGT + FA<JFJJJJJJJJJJJJJJJJJJJJJFFJJJFJJJJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:9699:1384 1:N:0:NAGAGG TGGGTGTGTAGGGGTGTAGTTTAATGGTTGGGGTGTTGTAAA + FFJJFJJJJFJJJJAFFJJJFJJJJJJFJJJJJ<JJJJJJJJ @K00316:163:HTYN2BBXX:5:1101:9800:1384 1:N:0:NAGAGG CGGTTAATAGTTGTAATTTTATTTTTTGTTTGTTTTTAAGGG + AFFJJJJJJJFFJJJJJJJJJJJJJJJJFJJJAJ-FJJJJJJ

khuang28jhu commented 6 years ago

The seed length of 20 is too large for read length around 50. In general, at least for SNAP is concerned, the seed length of these hash-based methods should be 1/4 of the average read length.

sbaheti commented 6 years ago

i tried reducing the seed but minimum seed length i can use is 16 according to SNAP. In this case it seems i can't use bsseker-3 because of shorter read length.